SILENT Fc VARIANTS OF ANTI-CD40 ANTIBODIES

ABSTRACT

The present invention relates to silent Fc variants of anti-CD40 antibodies and compositions and methods of use of said antibodies for treating pathological disorders such as autoimmune and inflammatory disorders and/or for preventing or reducing the risk of graft rejection in transplantation.

This application claims benefit under 35 U.S.C. §119(e) of U.S.application Ser. No. 13/295,141 U.S. Provisional Application No.61/413,567, filed Nov. 15, 2010, the content of which is incorporatedherein by reference in its entirety.

The present invention relates to silent constant fragment (Fc) variantsof anti-CD40 antibodies and compositions and methods of use of saidantibodies for treating pathological disorders such as autoimmune andinflammatory disorders and/or for preventing or reducing the risk ofgraft rejection in transplantation.

Despite availability of several immunosuppressive treatments forautoimmune diseases, there remains a large unmet need for moreefficacious and safer drugs in a large fraction of the patientpopulation. For example, despite the reported efficacy of B celldepleting/inhibiting therapies like Rituximab and Belimumab inrheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome,and multiple sclerosis, these therapies are only effective in a portionof diseased individuals, and, with Rituximab, with an accompanying riskof progressive multifocal leukoencephalopathy. Further, multiple otherleukocyte cell types are often involved in the pathology of theseautoimmune diseases such as macrophages, dendritic cells and T cells,therefore therapeutic intervention targeting additional cell types orkey immunological pathways that would inhibit their function couldprovide benefit. Given the multiple immunologically relevant roles ofCD40-CD154 in the activation and function or these cell types, it islikely that an anti-CD40 antibody would confer therapeutic benefit topatients suffering autoimmune diseases outlined above beyond thatcurrently provided by current therapies. Further, the central role forCD40-CD154 interactions in intestinal inflammatory disorders such asCrohn's disease and ulcerative colitis, and mechanistic links of theCD40 pathway to pathology in more rare disorders such as autoimmunevasculitis, pemphigus vulgaris, and ITP also highlights the potential ofanti-CD40 antibodies in these indications.

The currently available immunosuppressants used after solid organtransplantation provide excellent short-tem efficacy. Acute rejectionswithin the de novo period are observed in 5%-20% of the recipients(depending on organ, patient population, and regimen) and the proportionof grafts lost to acute rejection within the de novo period is below 5%for any setting. Currently the key unmet need is the tolerability ofimmunosuppression with patient and graft survival in the long term.After renal transplant, 33% patients die and/or lose their graft within5 years; the average age of death of transplant recipient is 58 years.Calcineurin inhibitors (CNI) remain the mainstay of immunosuppressivetherapy for the vast majority of transplant patients. Whilenephrotoxicity and cardiovascular morbidity associated with CNIs is oneof the drivers of chronic allograft nephropathy as well as patient deathwith a functioning graft, alternative primary immunosuppression have notbeen able to replace CNIs. Overall, there is still room for improvementin long-term transplant immunosuppression. B-cell mediated immunologicaldamage of transplanted kidneys may contribute to poor long-term outcomesand the need for new agents to target B-cell rejection is increasinglyrecognized by the medical community.

Chir12.12 is a fully humanized, non-agonist anti-CD40 antibody (IgG1,kappa) that blocks CD154 (also known as CD40 ligand; CD40L)-mediatedleukocyte activation and can mediate antibody-dependent cellularcytotoxicity (ADCC) of human leukocytes and B cell lymphomas in vitro(see WO2006/073443). WO2005/044306 also describes anti-CD40 antagonistantibodies, including Chir12.12 for use in particular in the treatmentof autoimmune and inflammatory disorders. Further Chir12.12 is effectivein delaying kidney allograft rejection when dosed as a monotherapy inMacaca fascicularis (Cynomolgus monkeys) [Li et al. (2008)Transplantation; 86 (1):10-15]. However, Chir12.12 can also mediatedepletion of peripheral B cells in non human primates (NHPs).

Anti-CD40 mAbs with silenced ADCC activity are predicted to have animproved safety profile relative to the parental anti-CD40 antibodies,and in particular may be more suitable for non-oncologic indications,such as autoimmune diseases and use in a transplant setting.

The present invention therefore provides Fc silent anti-CD40 monoclonalantibodies that retain the non-agonistic, CD40L blocking attributes ofthe parental anti-CD40 antibody Chir12.12.

In particular, the invention provides an isolated antibody or a proteincomprising an antigen-binding portion of an antibody directed againstthe target CD40 polypeptide (SEQ ID NO:28), characterized in that saidantibody or protein

-   -   a) binds to CD40 polypeptide with a K_(D) of 10 nM or less, and,    -   b) comprises a silent IgG Fc region.

In one embodiment, said antibody or protein inhibits CD40L inducedsignalling with an IC₅₀ of 50 ng/ml or less.

In another embodiment, the isolated antibody or protein according to theinvention has no or low agonist activity with respect to CD40signalling.

In another embodiment, the antibody or protein according to the presentinvention comprises a silent IgG Fc region selected from the groupconsisting of the amino acid sequence of SEQ ID NO:17, SEQ ID NO:18 orSEQ ID NO:19.

In another embodiment, the isolated antibody or protein of the inventioncomprises heavy chain (V_(H)) and light chain (V_(L)) variable regionshaving at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100 percentsequence identity to V_(H) of Chir12.12 (SEQ ID NO:9) and V_(L) ofChir12.12 antibody (SEQ ID NO:10) respectively.

Specific examples of the antibodies according to the invention are

-   -   mAb1 comprising heavy chain amino acid sequence of SEQ ID NO: 11        and light chain amino acid sequence of SEQ ID NO:12,    -   mAb2 comprising heavy chain amino acid sequence of SEQ ID NO: 13        and light chain amino acid sequence of SEQ ID NO:14, or,    -   mAb3 comprising heavy chain amino acid sequence of SEQ ID NO: 15        and light chain amino acid sequence of SEQ ID NO:16.

The isolated antibody or protein according to the invention may be usedas a medicament. In particular, they are suitable for use in thetreatment of autoimmune disorders, inflammatory disorders and/or inpreventing or reducing the risk of graft rejection in transplantation.

The isolated antibody or protein according to the invention may be usedin particular in the treatment of Multiple Sclerosis, Systemic LupusErythematosus, Sjögren's syndrome, Rheumatoid Arthritis, transplantrejection and graft-versus-host disease.

The invention also relates to pharmaceutical compositions comprising theabove antibodies or proteins according to the invention, in combinationwith at least a pharmaceutically acceptable excipient, diluent orcarrier. Said pharmaceutical compositions may additionally compriseother active ingredients.

The invention also relates to a lyophilisate or a liquid formulation ofan antibody or protein according to the invention.

The invention further relates to the isolated nucleic acid encoding theantibody or protein according to the invention and the correspondingcloning or expression vector comprising at least one nucleic acidselected from the group consisting of SEQ ID NOs: 22 to 27.

The invention also relates to a host cell comprising one or more cloningor expression vectors as defined above.

The invention further provides a process for the production of anantibody or a protein of the invention, comprising culturing the hostcell as defined above, purifying and recovering said antibody orprotein.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” or “signaling activity” refers to abiochemical causal relationship generally initiated by a protein-proteininteraction such as binding of a growth factor to a receptor, resultingin transmission of a signal from one portion of a cell to anotherportion of a cell. In general, the transmission involves specificphosphorylation of one or more tyrosine, serine, or threonine residueson one or more proteins in the series of reactions causing signaltransduction. Penultimate processes typically include nuclear events,resulting in a change in gene expression.

The term CD40 refers to human CD40, for example as defined in SEQ ID NO:28, unless otherwise described.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragments (i.e., “antigen-binding portion”) orsingle chains thereof.

A naturally occurring “antibody” is a glycoprotein comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as V_(H)) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, C_(L). The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antigenportion”), as used herein, refers to full length or one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., a portion of CD40). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and CH1 domains; a F(ab)₂ fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment consisting of the V_(H) and CH1 domains; a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), whichconsists of a V_(H) domain; and an isolated complementarity determiningregion (CDR), or any fusion proteins comprising such antigen-bindingportion.

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single chain protein in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc.Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

As used herein, the term “IgG Fc region” is used to define theC-terminal region of an immunoglobulin heavy chain, including nativesequence Fc region and variant Fc regions. The human IgG heavy chain Fcregion is generally defined as comprising the amino acid residue fromposition C226 or from P230 to the carboxyl-terminus of the IgG antibody.The numbering of residues in the Fc region is that of the EU index ofKabat. The C-terminal lysine (residue K447) of the Fc region may beremoved, for example, during production or purification of the antibody.Accordingly, a composition of antibodies of the invention may compriseantibody populations with all K447 residues removed, antibodypopulations with no K447 residues removed, and antibody populationshaving a mixture of antibodies with and without the K447 residue.

An “isolated antibody”, as used herein, refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds toCD40 is substantially free of antibodies that specifically bind to otherantigens than CD40). An isolated antibody that specifically binds toCD40 may, however, have cross-reactivity to other antigens, such as CD40molecules from other (non-human) species. Moreover, an isolated antibodymay be substantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “humanized antibody”, as used herein, is intended to includeantibodies that contain minimal sequence derived from non-humanimmunoglobulin sequences. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from ahypervariable region (also known as complementarity determining regionor CDR) of the recipient are replaced by residues from a hypervariableregion of a non-human species (donor antibody) such as mouse, rat,rabbit, or non-human primate having the desired specificity, affinityand capacity. The phrase “complementarity determining region” refers toamino acid sequences which together define the binding affinity andspecificity of the natural Fv region of a native immunoglobulin bindingsite. See, e.g. Chothia et al. (1987) J. Mol. Biol. 196:901-917: Kabatet al. (1991) US Dept. of Health and Human Services, NIH Publication No.91-3242). The phrase “constant region” refers to the portion of theantibody molecule that confers effector functions. In previous work,directed towards producing non-immunogenic antibodies for use in therapyof human disease, mouse constant regions were substituted by humanconstant regions. The constant regions of the subject humanizedantibodies were derived from human immunoglobulins. Humanization can beperformed following the method of Winter and co-coworkers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327:Verhoeyen et al. (1988) Science 239: 1534-1536), by substituting rodentand mutant rodent CDRs or CDR sequences for the corresponding sequencesof human antibody. In some instances, residues within the frameworkregions of one or more variable regions of the human immunoglobulin arereplaced by corresponding non-human residues (see, for example, U.S.Pat. Nos. 5,585,089; 5,693,761; 5,693,762; and 6,180,370). Furthermore,humanized antibodies may comprise residues that are not found in therecipient antibody or in the donor antibody. The humanized antibody ofthe invention will also comprise at least a portion of an immunoglobulinconstant region (Fc). Typically, that of a human immunoglobulin and inthe present case, a silent Fc IgG region.

The antibodies of the invention may include amino acid residues notencoded by human sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo). Inparticular, the term “humanized antibody” include antibodies thatcomprise a silent variant of Fc IgG region.

The term “humanized monoclonal antibody” refers to antibodies displayinga single binding specificity which have variable regions in which thevariable regions are humanized from non-human sequences.

The term “recombinant antibody”, as used herein, includes all human orhumanized antibodies that are prepared, expressed, created or isolatedby recombinant means, such as antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human or humanized antibody, e.g., froma transfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human or humanized antibodies have variable regions in whichthe framework and CDR regions may be derived from human germlineimmunoglobulin sequences. In certain embodiments, however, suchrecombinant human or humanized antibodies can be subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe V_(H) and V_(L) regions of the recombinant antibodies are sequencesthat, while derived from and related to human germline V_(H) and V_(L)sequences, may not naturally exist within the human antibody germlinerepertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM, IgE,IgG such as IgG1 or IgG4) that is provided by the heavy chain constantregion genes. Different isotypes have different effector function. Forexample, wild type human IgG1 and IgG3 isotypes mediateantibody-dependent cell-mediated cytotoxicity (ADCC) activity.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” and “an antibody directed against an antigen”are used interchangeably herein with the term “an antibody which bindsspecifically to an antigen”.

As used herein, an antibody or a protein that “specifically binds toCD40 polypeptide” is intended to refer to an antibody or protein thatbinds to human CD40 polypeptide with a K_(D) of 100 nM or less, 10 nM orless, 1 nM or less.

An antibody that “cross-reacts with an antigen other than CD40” isintended to refer to an antibody that binds that antigen with a K_(D) of1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less. An antibodythat “does not cross-react with a particular antigen” is intended torefer to an antibody that binds to that antigen, with a K_(D) of 100 nMor greater, or a K_(D) of 1 μM or greater, or a K_(D) of 10 μM orgreater. In certain embodiments, such antibodies that do not cross-reactwith the antigen exhibit essentially undetectable binding against theseproteins in standard binding assays.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction.

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e. K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A method for determining the K_(D) of anantibody is by using surface plasmon resonance, or using a biosensorsystem such as a Biacore® system.

As used herein, the term “Affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with the antigen at numeroussites; the more interactions, the stronger the affinity.

As used herein, the term “Avidity” refers to an informative measure ofthe overall stability or strength of the antibody-antigen complex. It iscontrolled by three major factors: antibody epitope affinity; thevalence of both the antigen and antibody; and the structural arrangementof the interacting parts. Ultimately these factors define thespecificity of the antibody, that is, the likelihood that the particularantibody is binding to a precise antigen epitope.

As used herein, the term “CD40 antagonist” is intended to refer to anantibody or protein that inhibits CD40 induced signaling activity in thepresence of CD40L in a human cell assay such as the CD40L-mediated PBMCproliferation assay. Such assay is described in more detail in theexamples below. In some embodiments, the antibodies or proteins of theinvention inhibit CD40L induced signaling with an IC50 of 500 ng/ml orless, preferably with an IC50 of 50 ng/ml or less, for example with anIC50 of 20 ng/ml or less, as measured in CD40L-mediated PBMCproliferation assay.

As used herein, an antibody with “no agonist activity” is intended torefer to an antibody that does not significantly increase CD40 mediatedsignaling activity in the absence of CD40L in a cell-based assay, suchas the CD40L-mediated PBMC proliferation assay. Such assay is describedin more details in the examples below.

As used herein, the term “ADCC” or “antibody-dependent cellularcytotoxicity” activity refers to cell depleting activity. ADCC activitycan be measured by the ADCC assay as described in more details in theExamples below.

As used herein, the term “silent” antibody refers to an antibody thatexhibits no or low ADCC activity as measured in an ADCC assay asdescribed in the Examples.

In one embodiment, the term “no or low ADCC activity” means that thesilent antibody exhibits an ADCC activity that is below 50% specificcell lysis, for example below 10% specific cell lysis as measured in theADCC assay as described in the Examples. No ADCC activity means that thesilent antibody exhibits an ADCC activity (specific cell lysis) that isbelow 1%. In a specific embodiment, a silent antibody according to theinvention does not exhibit any significant ADCC activity as measured inan ADCC assay as described in the Examples.

Silenced effector functions can be obtained by mutation in the Fc regionof the antibodies and have been described in the art: LALA and N297A(Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691); andD265A (Baudino et al., 2008, J. Immunol. 181: 6664-69; Strohl, W.,supra). Examples of silent Fc IgG1 antibodies comprise the so-calledLALA mutant comprising L234A and L235A mutation in the IgG1 Fc aminoacid sequence. Another example of a silent IgG1 antibody comprises theD265A mutation. Another silent IgG1 antibody comprises the N297Amutation, which results in aglycosylated/non-glycosylated antibodies.

As used herein, the term “selectivity” for an antibody or protein of theinvention refers to an antibody or protein that binds to a certaintarget polypeptide but not to closely related polypeptides.

As used herein, the term “high affinity” for an antibody refers to anantibody having a K_(D) of 1 nM or less for a target antigen. As usedherein, the term “subject” includes any human or nonhuman animal.

The term “nonhuman animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dogs, cats, horses,cows, chickens, amphibians, reptiles, etc.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a cell of Trichoderma, a ChineseHamster Ovary cell (CHO) or a human cell. The optimized nucleotidesequence is engineered to retain completely or as much as possible theamino acid sequence originally encoded by the starting nucleotidesequence, which is also known as the “parental” sequence. The optimizedsequences herein have been engineered to have codons that are preferredin CHO mammalian cells, however optimized expression of these sequencesin other eukaryotic cells is also envisioned herein. The amino acidsequences encoded by optimized nucleotide sequences are also referred toas optimized.

As used herein, the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity=# of identical positions/total # of positions×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm, as described below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17, 1988) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. Alternatively, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The percent identity between two nucleotide amino acid sequences mayalso be determined using for example algorithms such as the BLASTNprogram for nucleic acid sequences using as defaults a word length (W)of 11, an expectation (E) of 10, M=5, N=4, and a comparison of bothstrands.

Recombinant Antibodies

Antibodies of the invention include the humanized recombinant antibodiesmAb1-mAb3, isolated and structurally characterized by their full lengthheavy and light chain amino acid sequences as described in the Table 1below:

TABLE 1 Full length heavy and light chain amino acid sequences ofmAb1-mAb3 Full Length Heavy Chain Full Length Light Chain Antibody Aminoacid sequence Amino acid sequence mAb1 SEQ ID NO: 11 SEQ ID NO: 12 mAb2SEQ ID NO: 13 SEQ ID NO: 14 mAb3 SEQ ID NO: 15 SEQ ID NO: 16

The corresponding variable regions, V_(H) and V_(L) amino acid sequencesof such isolated antibodies mAb1-mAb3 of the invention all derived fromthe same antibody Chir12.12 previously described for example inWO2006/073443 and consisting of V_(H) amino acid sequence of SEQ ID NO:7and V_(L) amino acid sequence of SEQ ID NO:8.

One important difference of the antibodies of the invention compared tooriginal CHIR12.12 is that they have an Fc region, consisting of asilent Fc IgG region, for example silent Fc IgG1 region.

In particular, Table 2 summarizes the modification of Fc IgG1 regionperformed to obtain the antibodies mAb1-mAb3 as compared to originalCHIR12.12 antibody.

TABLE 2 Modification of Fc IgG1 region to obtain mAb1-mAb3 Modificationof Fc IgG1 Fc region Antibody region Amino acid sequence mAb1 N297A SEQID NO: 17 mAb2 D265A SEQ ID NO: 18 mAb3 L234A/L235A SEQ ID NO: 19

Other antibodies of the invention include those having amino acids thathave been mutated by amino acid deletion, insertion or substitution, yethave at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100 percent identitywith the V_(H) and V_(L) regions of CHIR12.12, respectively SEQ ID NO:7and SEQ ID NO:8 and comprising a silent IgG Fc region, for example asilent IgG1 Fc region.

In some embodiments, the antibody of the invention is a mutant variantof any one of mAb1-mAb3, wherein said mutant variant antibody includemutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 aminoacids have been mutated by amino acid deletion, insertion orsubstitution in the V_(H) and V_(L) regions when compared with the V_(H)and V_(L) regions of CHIR12.12, respectively SEQ ID NO:7 and SEQ ID NO:8and retaining the same constant regions as mAb1, mAb2 or mAb3.

Full length light and heavy chain nucleotide coding sequences ofmAb1-mAb3 are shown in Table 3 below.

TABLE 3 Full length heavy and light chain DNA coding sequences FullLength Heavy Chain Full Length Light Chain Antibody DNA coding sequenceDNA coding sequence mAb1 SEQ ID NO: 22 SEQ ID NO: 23 mAb2 SEQ ID NO: 24SEQ ID NO: 25 mAb3 SEQ ID NO: 26 SEQ ID NO: 27

Other nucleic acids encoding antibodies of the invention include nucleicacids that have been mutated by nucleotide deletion, insertion orsubstitution, yet have at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or100 percent identity to the V_(H) and V_(L) corresponding coding regionsof CHIR12.12, as depicted in the sequences described for example in SEQID NO:20 and SEQ ID NO:21 respectively and comprising a coding sequenceof a silent IgG Fc region, for example, a silent IgG1 Fc region.

In some embodiments, it includes variant nucleic acids wherein no morethan 1, 2, 3, 4 or 5 nucleotides have been changed by nucleotidedeletion, insertion or substitution in the V_(H) and V_(L) codingregions with the V_(H) and V_(L) coding regions depicted in thesequences described for example in SEQ ID NO:20 and SEQ ID NO:21respectively, and retaining the same coding sequences of the constantregions as mAb1, mAb2 or mAb3 corresponding coding sequences.

Homologous Antibodies

In addition to the recombinant antibodies of the invention, mAb1-mAb3,the invention also encompasses homologous antibodies or proteinsretaining the desired functional properties of mAb1-mAb3 antibodies.

In particular, said homologous antibodies or proteins according to theinvention are antibodies or proteins comprising an antigen-bindingportion of an antibody directed against a target CD40 polypeptide (SEQID NO:28), characterized in that said antibody or protein

-   -   a) binds to CD40 polypeptide with a K_(D) of 10 nM or less, and,    -   b) comprises a silent IgG Fc region        and wherein said homologous antibodies or proteins retain the        desired functional properties of the original mAb1-mAb3        antibodies.

Desired functional properties of the original mAb1-mAb3 antibodies maybe selected from one ore more of the following properties:

-   -   (i) it specifically binds to CD40, for example, a K_(D) being        100 nM or less, 10 nM or less, or 1 nM or less, as measured in        the Biacore assay;    -   (ii) it is a CD40 antagonist, for example, it inhibits CD40L        induced signaling as measured in CD40L-mediated PBMC        proliferation assay;    -   (iii) it exhibits no or low agonist activity, as measured in a        CD40L-mediated PBMC proliferation assay;    -   (iv) it cross-reacts with Cynomolgus monkey CD40 polypeptide;    -   (v) it has no or low ADCC activity; and,    -   (vi) it has suitable properties for drug development.

In one specific embodiment, said homologous antibodies or proteinsaccording to the invention comprise a silent IgG1 Fc region, forexample, a silent IgG1 Fc region selected from the group consisting ofSEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

In one specific embodiment, the invention relates to an antibody orprotein which has variable region heavy and light chain nucleotidesequences, or variable region heavy and light chain amino acidsequences, or all 6 CDRs amino acid sequences or nucleotide codingsequences that are homologous to the corresponding amino acid ornucleotide sequences of the antibodies mAb1-mAb3 described above, inparticular in Table 1, and said antibody or protein comprise a silentIgG Fc region selected from the group consisting of the Fc region ofmAb1 (SEQ ID NO:17), the Fc region of mAb2 (SEQ ID NO:18) and the Fcregion of mAb3 (SEQ ID NO:19), wherein said homologous antibody orprotein specifically binds to CD40, and the antibody or protein exhibitsthe following functional properties: it is a CD40 antagonist, itexhibits no or low agonist activity, and it has no or low ADCC activity.

For example, the invention relates to antibodies or proteins homologousto mAb1-mAb3, comprising a silent IgG Fc region selected from the groupconsisting of the Fc region of mAb1 (SEQ ID NO:17), the Fc region ofmAb2 (SEQ ID NO:18) and the Fc region of mAb3 (SEQ ID NO:19), andcomprising a variable heavy chain (V_(H)) and a variable light chain(V_(L)) sequences where the CDR sequences share at least 60, 70, 90, 95,96, 97, 98, 99 or 100 percent sequence identity to the corresponding CDRsequences of mAb1-mAb3, respectively SEQ ID NOs:1-6, wherein saidhomologous antibody or protein specifically binds to CD40, and thehomologous antibody or protein exhibits the following functionalproperties: it is a CD40 antagonist, it exhibits no or low agonistactivity, and it has no or low ADCC activity.

In a related specific embodiment, the homologous antibody or protein

-   -   a) binds to CD40 with a K_(D) of 1 nM or less;    -   b) inhibits CD40L induced signaling with an IC50 of 50 ng/ml or        less as measured in CD40L-mediated PBMC proliferation assay        described in the Examples;    -   c) has no or low agonist activity as measured in a bioassay such        as CD40L-mediated PBMC proliferation assay as described in the        Examples; and,    -   d) has no or low ADCC activity.

The invention further relates to antibodies or proteins homologous tomAb1-mAb3, comprising a silent IgG Fc region selected from the groupconsisting of the Fc region of mAb1 (SEQ ID NO:17), the Fc region ofmAb2 (SEQ ID NO:18) and the Fc region of mAb3 (SEQ ID NO:19), andcomprising a variable heavy chain (V_(H)) and a variable light chain(V_(L)) sequences which share at least 80, 90, 95, 96, 97, 98, 99 or 100percent sequence identity to the corresponding (V_(H)) and (V_(L))sequences of mAb1-mAb3, respectively SEQ ID NO:7 and SEQ ID NO:8,wherein said homologous antibody or protein specifically binds to CD40,and the antibody or protein exhibits the following functionalproperties: it is a CD40 antagonist, it exhibits no or low agonistactivity, and it has no or low ADCC activity.

In a related specific embodiment, said homologous antibody or protein

-   -   a) binds to CD40 with a K_(D) of 1 nM or less;    -   b) inhibits CD40L induced signaling with an IC50 of 50 ng/ml or        less as measured in CD40L-mediated PBMC proliferation assay        described in the Examples;    -   c) has no or low agonist activity as measured in a bioassay such        as CD40L-mediated PBMC proliferation assay described in the        Examples; and,    -   d) has no or low ADCC activity.

In another example, the invention relates to antibodies or proteinshomologous to mAb1-mAb3 comprising a silent IgG Fc region selected fromthe group consisting of the Fc region of mAb1 (SEQ ID NO:17), the Fcregion of mAb2 (SEQ ID NO:18) and the Fc region of mAb3 (SEQ ID NO:19),and wherein: the variable heavy and light chains are encoded by anucleotide sequence that is at least 80%, at least 90%, at least 95%, or100% identical to the corresponding coding nucleotide sequence of thevariable heavy and light chains of mAb1-mAb3, wherein said homologousantibody or protein specifically binds to CD40, and the antibody orprotein exhibits the following functional properties: it is a CD40antagonist, it exhibits no or low agonist activity, and it has no or lowADCC activity.

In a related specific embodiment, said homologous antibody or protein

-   -   a) binds to CD40 with a K_(D) of 1 nM or less;    -   b) inhibits CD40L induced signaling with an IC50 of 50 ng/ml or        less as measured in CD40L-mediated PBMC proliferation assay        described in the Examples;    -   c) has no or low agonist activity as measured in a bioassay such        as CD40L-mediated PBMC proliferation assay described in the        Examples; and,    -   d) has no or low ADCC activity.

Antibodies with mutant amino acid sequences can be obtained bymutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of thecoding nucleic acid molecules, followed by testing of the encodedaltered antibody for retained function (i.e., the functions set forthabove) using the functional assays described in the Examples below.

In certain embodiments, the antibodies or proteins homologous tomAb1-mAb3 as described above have conservative sequence modifications.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid substitutions in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), non-polar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family, and the altered antibody can be tested for retainedfunction using the functional assays described herein.

Modifications can be introduced into an antibody of the invention bystandard techniques known in the art, such as site-directed mutagenesisand PCR-mediated mutagenesis.

Nucleic Acid Molecules Encoding Antibodies or Proteins of the Invention

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibodies or proteins of the invention as described above.

Examples of light and heavy chains nucleotide sequences of any one ofmAb1 to mAb3 can be derived from the Table 3 (showing the entirenucleotide coding sequences of heavy and light chains of mAb1 to mAb3).

Other examples of light and heavy chains nucleotide sequences accordingto the invention are any sequence coding for the full length heavyand/or light amino acid sequences of mAb1, mAb2 or mAb3 as described inTable 1.

The invention also pertains to nucleic acid molecules that derive fromthe latter sequences having been optimized for protein expression inmammalian cells, for example, CHO cell lines.

The nucleic acids may be present in whole cells, in a cell lysate, ormay be nucleic acids in a partially purified or substantially pure form.A nucleic acid is “isolated” or “rendered substantially pure” whenpurified away from other cellular components or other contaminants,e.g., other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis and others well known in the art. See, F.Ausubel, et al., ed. 1987 Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York. A nucleic acid of theinvention can be, for example, DNA or RNA and may or may not containintronic sequences. In an embodiment, the nucleic acid is a cDNAmolecule. The nucleic acid may be present in a vector such as a phagedisplay vector, or in a recombinant plasmid vector.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. Once DNA fragments encoding, for example, V_(H) andV_(L) segments are obtained, these DNA fragments can be furthermanipulated by standard recombinant DNA techniques, for example toconvert the variable region genes to full-length antibody chain genes,to Fab fragment genes or to an scFv gene. In these manipulations, aV_(L)- or V_(H)-encoding DNA fragment is operatively linked to anotherDNA molecule, or to a fragment encoding the antibody constant region ofmAb1-mAb3 comprising Fc region as defined in SEQ ID NOs:17-19.

The term “operatively linked”, as used in this context, is intended tomean that the two DNA fragments are joined in a functional manner, forexample, such that the amino acid sequences encoded by the two DNAfragments remain in-frame, or such that the protein is expressed undercontrol of a desired promoter.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., et al., 1991 Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region may be selected amongIgG1 isotypes comprising Fc region as defined in SEQ ID NOs:17-19.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as to a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al., 1991 Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or a lambda constant region.

Generation of Transfectomas Producing Monoclonal Antibodies

Antibodies or proteins of the invention can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, DNAs encoding partial orfull-length light and heavy chains can be obtained by standard molecularbiology or biochemistry techniques (e.g., DNA chemical synthesis, PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector.

The antibody genes are inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes by inserting them into expression vectorsalready encoding heavy chain constant and light chain constant regionsof the desired sequence corresponding to said constant regions of mAb1,mAb2 or mAb3 such that the V_(H) segment is operatively linked to theC_(H) segment(s) within the vector and the V_(L) segment is operativelylinked to the C_(L) segment within the vector. Additionally oralternatively, the recombinant expression vector can encode a signalpeptide that facilitates secretion of the antibody chain from a hostcell. The antibody chain gene can be cloned into the vector such thatthe signal peptide is linked in frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. 1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Regulatory sequences for mammalian host cell expression includeviral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., theadenovirus major late promoter (AdMLP)), and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or P-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRa promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al., 1988 Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains are transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. It is theoretically possible toexpress the antibodies of the invention in either prokaryotic oreukaryotic host cells. Expression of antibodies in eukaryotic cells, forexample mammalian host cells, yeast or filamentous fungi, is discussedbecause such eukaryotic cells, and in particular mammalian cells, aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active antibody.

In one specific embodiment, a cloning or expression vector according tothe invention comprises either at least one of the following codingsequences (a)-(c), operatively linked to suitable promoter sequences:

(a) SEQ ID NO:22 and SEQ ID NO:23 encoding respectively the full lengthheavy and light chains of mAb1;(b) SEQ ID NO:24 and SEQ ID NO:25 encoding respectively the full lengthheavy and light chains of mAb2; or,(c) SEQ ID NO:26 and SEQ ID NO:27 encoding respectively the full lengthheavy and light chains of mAb3.

Mammalian host cells for expressing the recombinant antibodies of theinvention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHOcells, described Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA77:4216-4220 used with a DH FR selectable marker, e.g., as described inR. J. Kaufman and P. A. Sharp, 1982 Mol. Biol. 159:601-621), CHOK1 dhfr+cell lines, NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another expression system is the GS geneexpression system shown in PCT Publications WO 87/04462, WO 89/01036 andEP 0 338 841.

When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods (See for example Abhinav et al. 2007,Journal of Chromatography 848: 28-37).

In one specific embodiment, the host cell of the invention is a hostcell transfected with an expression vector having the coding sequencesselected from the group consisting of (a)-(c) suitable for theexpression of mAb1-mAb3 respectively, operatively linked to suitablepromoter sequences:

(a) SEQ ID NO:22 and SEQ ID NO:23;

(b) SEQ ID NO:24 and SEQ ID NO:25; and,

(c) SEQ ID NO:26 and SEQ ID NO:27.

The latter host cells may then be further cultured under suitableconditions for the expression and production of an antibody of theinvention selected from the group consisting of mAb1-mAb3 respectively.

Bispecific Molecules

In another aspect, the present invention features bispecific ormultispecific molecules comprising an anti-CD40 antibody or protein ofthe invention. An antibody or protein of the invention can bederivatized or linked to another functional molecule, e.g., anotherpeptide or protein (e.g., another antibody or ligand for a receptor) togenerate a bispecific molecule that binds to at least two differentbinding sites or target molecules. The antibody of the invention may infact be derivatized or linked to more than one other functional moleculeto generate multi-specific molecules that bind to more than twodifferent binding sites and/or target molecules; such multi-specificmolecules are also intended to be encompassed by the term “bispecificmolecule” as used herein. To create a bispecific molecule of theinvention, an antibody or protein of the invention can be functionallylinked (e.g., by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other binding molecules, suchas another antibody, antibody fragment, peptide or binding mimetic, suchthat a bispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for CD40, for example,one antigen-binding portion of any one of mAb1-mAb3 and a second bindingspecificity for a second target epitope. For example, the second targetepitope is another epitope of CD40 different from the first targetepitope. Another example is a bispecific molecule comprising at leastone first binding specificity for CD40, for example, one antigen-bindingportion of any one of mAb1-mAb3 and a second binding specificity for anepitope within CD40.

Additionally, for the invention in which the bispecific molecule ismulti-specific, the molecule can further include a third bindingspecificity, in addition to the first and second target epitope.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, using methods knownin the art. For example, each binding-specificity of the bispecificmolecule can be generated separately and then conjugated to one another.When the binding specificities are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-l-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686;Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus, 1985 Behring Ins. Mitt. No. 78,118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al.,1987 J. Immunol. 139: 2367-2375). Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particular embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (REA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest.

Multivalent Antibodies

In another aspect, the present invention provides multivalent antibodiescomprising at least two identical or different antigen-binding portionsof the antibodies of the invention binding to CD40, for example,selected from antigen-binding portions of any one of mAb1-mAb3. In oneembodiment, the multivalent antibodies provide at least two, three orfour antigen-binding portions of the antibodies. The antigen-bindingportions can be linked together via protein fusion or covalent or noncovalent linkage. Alternatively, methods of linkage have been describedfor the bispecific molecules. Tetravalent compounds can be obtained forexample by cross-linking antibodies of the antibodies of the inventionwith an antibody that binds to the constant regions of the antibodies ofthe invention, for example the Fc or hinge region.

Methods of Therapy Using the Antagonist Anti-CD40 Antibodies of theInvention

Methods of the invention are directed to the use of the anti-CD40antibodies or proteins of the invention to treat subjects (i.e.,patients) having an autoimmune disease and/or inflammatory disease, or apredisposition to developing an autoimmune disease and/or inflammatorydisease, wherein the disease and/or inflammation is mediated byCD40L-mediated CD40 signaling on cells expressing the CD40 antigen.

Methods for detecting CD40 expression in cells are well known in the artand include, but are not limited to, PCR techniques,immunohistochemistry, flow cytometry, Western blot, ELISA, and the like.

The methods of the invention are especially useful for treatinginflammatory and/or autoimmune diseases wherein CD40L-mediated CD40stimulation is involved.

Inflammatory diseases are characterized by inflammation and tissuedestruction, or a combination thereof. “Inflammatory disease” includesany inflammatory immune-mediated process where the initiating event ortarget of the immune response involves non-self antigen (s), including,for example, alloantigens, xenoantigens, viral antigens, bacterialantigens, unknown antigens, or allergens.

Further, for purposes of the present invention, the term “inflammatorydisease(s)” includes “autoimmune disease(s)”. As used herein, the term“autoimmunity” is generally understood to encompass inflammatoryimmune-mediated processes involving “self” antigens. In autoimmunediseases, self antigen(s) trigger host immune responses.

Also, the present invention includes treatment of inflammationassociated with tissue transplant rejection. “Transplant rejection” or“graft rejection” refers to any host-mounted immune response against agraft including but not limited to HLA antigens, blood group antigens,and the like.

The invention can also be used to treat graft versus host disease, suchas that associated with bone marrow transplantation, for example. Insuch graft versus host disease, the donor bone marrow includeslymphocytes and cells that mature into lymphocytes. The donorslymphocytes recognize the recipient's antigens as non-self and mount aninflammatory immune response. Hence, as used herein, “graft versus hostdisease” or “graft versus host reaction” refers to any T cell mediatedimmune response in which donor lymphocytes react to the host's antigens.

The antagonist anti-CD40 antibodies or proteins described herein, forexample mAb1, mAb2 or mAb3, can be used in accordance with the methodsof the invention to treat autoimmune and/or inflammatory disordersincluding, but not limited to, systemic lupus erythematosus (SLE),discoid lupus, lupus nephritis, sarcoidosis, inflammatory arthritis,including juvenile arthritis, rheumatoid arthritis, psoriatic arthritis,Reiter's syndrome, ankylosing spondylitis, and gouty arthritis,rejection of an organ or tissue transplant, hyperacute, acute, orchronic rejection and/or graft versus host disease, multiple sclerosis,hyper IgE syndrome, polyarteritis nodosa, primary biliary cirrhosis,inflammatory bowel disease, Crohn's disease, celiac's disease(gluten-sensitive enteropathy), primary Sjögren's syndrome (pSS),autoimmune hepatitis, pernicious anemia, autoimmune hemolytic anemia,psoriasis, scleroderma, myasthenia gravis, autoimmune thrombocytopenicpurpura, autoimmune thyroiditis, Grave's disease, Hasimoto'sthyroiditis, immune complex disease, chronic fatigue, immune dysfunctionsyndrome (CFIDS), polymyositis and dermatomyositis, cryoglobulinemia,thrombolysis, cardiomyopathy, pemphigus vulgaris, pulmonary interstitialfibrosis, Type I and Type II diabetes mellitus, type 1, 2, 3, and 4delayed-type hypersensitivity, allergy or allergic disorders,unwanted/unintended immune responses to therapeutic proteins (see forexample, U. S. Patent Application No. US 2002/0119151 and Koren, et al.(2002) Curr. Pharm. Biotechnol. 3: 349-60), asthma, Churg-Strausssyndrome (allergic granulomatosis), atopic dermatitis, allergic andirritant contact dermatitis, urtecaria, IgE-mediated allergy,atherosclerosis, ANCA-associated Vasculitides, vasculitis, idiopathicinflammatory myopathies, hemolytic disease, Alzheimer's disease, chronicinflammatory demyelinating polyneuropathy, and the like.

Genetic ablation or pharmacological inhibition of the CD40-CD154 pathwayhas previously demonstrated therapeutic benefit in either the clinic orin preclinical models of SLE, pSS, ITP, MS, Crohn's disease, Pemphigusvulgaris, autoimmune vasculitis and RA (Law C L, Grewal I S. (2009).Adv. Exp. Med. Biol. 2009; 647:8-36); the medical need of which isdetailed below.

In preferred embodiments the anti-CD40 antibodies or proteins of theinvention are useful in treating: (i) systemic lupus erythematosus(lupus nephritis), preferably in providing effective steroid-sparingtherapies for induction and maintenance of remission, and prevention ofend-stage renal disease; (ii) primary Sjögren's syndrome, preferably inprevention of salivary and lacrimary gland destruction, and inductionand maintenance of remission of extraglandular manifestations; (iii)autoimmune thrombocytopenic purpura, preferably treatment of patientsrefractory to standard of care; (iv) ANCA-associated Vasculitides,preferably inducing and maintaining remission in patients refractory tocorticosteroids, and steroid-sparing treatment; (v) Pemphigus Vulgaris,preferably in induction and maintenance of remission in patientsrefractory to corticosteroids, and steroid-sparing treatment; (vi)Multiple Sclerosis, preferably in providing more effective treatmentsfor prevention of relapses and disability progression, and achievingdisease-free status; and (vii) Crohn's disease, preferably in providingmore effective therapies for maintenance of remission, and treatment ofpatients refractory to anti-TNF.

In some other embodiments, the anti-CD40 antibodies or proteins of theinvention are useful in treating pulmonary inflammation including butnot limited to lung graft rejection, asthma, sarcoidosis, emphysema,cystic fibrosis, idiopathic pulmonary fibrosis, chronic bronchitis,allergic rhinitis and allergic diseases of the lung such ashypersensitivity pneumonitis, eosinophilic pneumonia, bronchiolitisobliterans due to bone marrow and/or lung transplantation or othercauses, graft atherosclerosis/graft phlebosclerosis, as well aspulmonary fibrosis resulting from collagen, vascular, and autoimmunediseases such as rheumatoid arthritis, scleroderma and lupuserythematosus.

“Treatment” is herein defined as the application or administration of ananti-CD40 antibody or protein according to the invention, for example,mAb1, mAb2 or mAb3 antibody, to a subject, or application oradministration a pharmaceutical composition comprising said anti-CD40antibody or protein of the invention to an isolated tissue or cell linefrom a subject, where the subject has an autoimmune disease and/orinflammatory disease, a symptom associated with an autoimmune diseaseand/or inflammatory disease, or a predisposition toward development ofan autoimmune disease and/or inflammatory disease, where the purpose isto cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve,or affect the autoimmune disease and/or inflammatory disease, anyassociated symptoms of the autoimmune disease and/or inflammatorydisease, or the predisposition toward the development of the autoimmunedisease and/or inflammatory disease.

By “treatment” is also intended the application or administration of apharmaceutical composition comprising an anti-CD40 antibodies or proteinof the invention, for example, mAb1, mAb2 or mAb3 antibody, to asubject, or application or administration of a pharmaceuticalcomposition comprising said anti-CD40 antibody or protein of theinvention to an isolated tissue or cell line from a subject, where thesubject has an autoimmune disease and/or inflammatory disease, a symptomassociated with an autoimmune disease and/or inflammatory disease, or apredisposition toward development of an autoimmune disease and/orinflammatory disease, where the purpose is to cure, heal, alleviate,relieve, alter, remedy, ameliorate, improve, or affect the autoimmunedisease and/or inflammatory disease, any associated symptoms of theautoimmune disease and/or inflammatory disease, or the predispositiontoward the development of the autoimmune disease and/or inflammatorydisease.

By “anti-inflammatory activity” is intended a reduction or prevention ofinflammation. Therapy with at least one anti-CD40 antibody or proteinaccording to the invention causes a physiological response that isbeneficial with respect to treatment of an autoimmune disease and/orinflammatory disease, where the disease involves cells expressing theCD40 antigen. It is recognized that the methods of the invention may beuseful in preventing phenotypic change in cells such as proliferation,activation, and the like.

In accordance with the methods of the present invention, at least oneanti-CD40 antibody or protein of the invention as defined above hereinis used to promote a positive therapeutic response with respect totreatment or prevention of an autoimmune disease and/or inflammatorydisease.

By “positive therapeutic response” with respect to an autoimmune diseaseand/or inflammatory disease is intended an improvement in the disease inassociation with the anti-inflammatory activity of these antibodies orproteins, and/or an improvement in the symptoms associated with thedisease. That is, an anti-proliferative effect, the prevention offurther proliferation of the CD40-expressing cell, a reduction in theinflammatory response including but not limited to reduced secretion ofinflammatory cytokines, adhesion molecules, proteases, immunoglobulins(in instances where the CD40 bearing cell is a B cell), combinationsthereof, and the like, increased production of anti-inflammatoryproteins, a reduction in the number of autoreactive cells, an increasein immune tolerance, inhibition of autoreactive cell survival, and/or adecrease in one or more symptoms mediated by stimulation ofCD40-expressing cells can be observed. Such positive therapeuticresponses are not limited to the route of administration and maycomprise administration to the donor, the donor tissue (such as forexample organ perfusion), the host, any combination thereof, and thelike.

Clinical response can be assessed using screening techniques such asmagnetic resonance imaging (MRI) scan, x-radiographic imaging, computedtomographic (CT) scan, flow cytometry or fluorescence-activated cellsorter (FACS) analysis, histology, gross pathology, and blood chemistry,including but not limited to changes detectable by ELISA, RIA,chromatography, and the like. In addition to these positive therapeuticresponses, the subject undergoing therapy with the antagonist anti-CD40antibody or protein of the invention may experience the beneficialeffect of an improvement in the symptoms associated with the disease.

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of anti-CD40 antibody or protein of the inventionthat, when administered brings about a positive therapeutic responsewith respect to treatment of a subject with an autoimmune disease and/orinflammatory disease.

In some embodiments of the invention, a therapeutically effective doseof the anti-CD40 antibody or protein of the invention, for example,mAb1, mAb2 or mAb3 is in the range from 0.01 mg/kg to 40 mg/kg, from 3mg/kg to 20 mg/kg or from 7 mg/kg to 12 mg/kg. It is recognized that themethod of treatment may comprise a single administration of atherapeutically effective dose or multiple administrations of atherapeutically effective dose of the antagonist anti-CD40 antibody orprotein of the invention.

A further embodiment of the invention is the use of anti-CD40 antibodiesor proteins of the invention for diagnostic monitoring of protein levelsin tissue as part of a clinical testing procedure, e.g., to determinethe efficacy of a given treatment regimen. Detection can be facilitatedby coupling the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, P-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, or ³H.

The anti-CD40 antibodies or proteins of the invention, for example,mAb1, mAb2 or mAb3 can be used in combination with any known therapiesfor autoimmune and inflammatory diseases, including any agent orcombination of agents that are known to be useful, or which have beenused or are currently in use, for treatment of autoimmune andinflammatory diseases. Such therapies and therapeutic agents include,but are not limited to, surgery or surgical procedures (e.g.splenectomy, lymphadenectomy, thyroidectomy, plasmaphoresis,leukophoresis, cell, tissue, or organ transplantation, intestinalprocedures, organ perfusion, and the like), radiation therapy, therapysuch as steroid therapy and non-steroidal therapy, hormone therapy,cytokine therapy, therapy with dermatological agents (for example,topical agents used to treat skin conditions such as allergies, contactdermatitis, and psoriasis), immunosuppressive therapy, and otheranti-inflammatory monoclonal antibody therapy, and the like. In thismanner, the antagonist anti-CD40 antibodies or proteins described hereinare administered in combination with at least one other therapy,including, but not limited to, surgery, organ perfusion, radiationtherapy, steroid therapy, non-steroidal therapy, antibiotic therapy,antifungal therapy, hormone therapy, cytokine therapy, therapy withdermatological agents (for example, topical agents used to treat skinconditions such as allergies, contact dermatitis, and psoriasis),immunosuppressive therapy, other anti-inflammatory monoclonal antibodytherapy, combinations thereof, and the like.

Thus, where the combined therapies comprise administration of ananti-CD40 antibody or protein of the invention such as mAb1, mAb2 ormAb3 antibody, in combination with administration of another therapeuticagent, as with steroids as one example, the methods of the inventionencompass co-administration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder. Where the methods of the present invention comprise combinedtherapeutic regimens, these therapies can be given simultaneously, i.e.,the anti-CD40 antibody or protein of the invention is administeredconcurrently or within the same time frame as the other therapy (i.e.,the therapies are going on concurrently, but the anti-CD40 antibody orprotein of the invention is not administered precisely at the same timeas the other therapy). Alternatively, the anti-CD40 antibody of thepresent invention or protein of the invention may also be administeredprior to or subsequent to the other therapy. Sequential administrationof the different therapies may be performed regardless of whether thetreated subject responds to the first course of therapy to decrease thepossibility of remission or relapse.

In some embodiments of the invention, the anti-CD40 antibodies orproteins of the invention, for example mAb1, mAb2 or mAb3 antibody, areadministered in combination with immunosuppressive drugs oranti-inflammatory drugs, wherein the antibody or protein and thetherapeutic agent (s) may be administered sequentially, in either order,or simultaneously (i.e., concurrently or within the same time frame).Examples of suitable immunosuppressive drugs that can be administered incombination with the antagonistic anti-CD40 antibodies or proteins ofthe invention, for example mAb1, mAb2 or mAb3 antibody, include, but arenot limited to, methotrexate, cyclophosphamide, mizoribine,chlorambucil, cyclosporine, such as, for example, aerosolizedcyclosporine (see, U. S. Patent Application Publication No. US2002/0006901), tacrolimus (FK506; ProGrafrM), mycophenolate mofetil, andazathioprine (6-mercaptopurine), sirolimus (rapamycin), deoxyspergualin,leflunomide and its malononitriloamide analogs; and immunosuppressiveproteins, including, for example, anti-CTLA4 antibodies and Ig fusions,anti-B lymphocyte stimulator antibodies (e.g., LYMPHOSTAT-BTM) and Igfusions (BLyS-Ig), anti-CD80 antibodies and etanercept (Enbrel®), aswell as anti-T cell antibodies such as anti-CD3 (OKT3), anti-CD4, andthe like. Examples of suitable anti-inflammatory agents include, but arenot limited to, corticosteroids such as, for example, clobetasol,halobetasol, hydrocortisone, triamcinolone, betamethasone, fluocinole,fluocinonide, prednisone, prednisolone, methylprednisolone;non-steroidal anti-inflammatory drugs (NSAIDs) such as, for example,sulfasalazine, medications containing mesalamine (known as 5-ASAagents), celecoxib, diclofenac, etodolac, fenprofen, flurbiprofen,ibuprofen, ketoprofen, meclofamate, meloxicam, nabumetone, naproxen,oxaprozin, piroxicam, rofecoxib, salicylates, sulindac, and tolmetin;phosphodiesterase-4 inhibitors, anti-inflammatory antibodies such asadalimumab (HUMERA®, a TNF-α antagonist) and infliximab (Remicade, aTNF-α antagonist), and the like. Also included are immune modulatingagents of current or potential use in treating autoimmune disease, suchas thalidomide or its analogs such as lenalidomide.

Transplant rejection and graft versus host disease can be hyperacute(humoral), acute (T cell mediated), or chronic (unknown etiology), or acombination thereof. Thus, the anti-CD40 antibodies or proteins of theinvention, for example mAb1, mAb2 or mAb3 antibody, are used in someembodiments to prevent and/or ameliorate rejection and/or symptomsassociated with hyperacute, acute, and/or chronic transplant rejectionof any tissue, including, but not limited to, liver, kidney, pancreas,pancreatic islet cells, small intestine, lung, heart, corneas, skin,blood vessels, bone, heterologous or autologous bone marrow, and thelike. Graft tissues may be obtained from any donor and transplanted intoany recipient host, and thus the transplant procedure may comprisetransplanting animal tissue to humans (e.g. xenografts), transplantingtissue from one human to another human (e.g. allografts), and/ortransplanting tissue from one part of a human's body to another (e.g.autografts).

Treatment with the antibodies or proteins of the invention may alsoreduce transplantation sequelae such as fever, anorexia, hemodynamicabnormalities, leukopenia, white cell infiltration of the transplantedorgan/tissue, as well as opportunistic infections.

In some embodiments, the anti-CD40 antibodies or proteins of theinvention, for example mAb1, mAb2 or mAb3 antibody, may be used alone orin combination with immunosuppressive drugs to treat and/or preventtransplant rejection such as hyperacute, acute, and/or chronic rejectionand/or graft versus host disease.

Thus, in some embodiments where the anti-CD40 antibodies or proteins ofthe invention are used to treat graft rejection, the antibodies, forexample mAb1, mAb2 or mAb3 antibody, may be used in combination withsuitable immunosuppressive drugs, including, but not limited, tomethotrexate; cyclophosphamide; mizoribine; chlorambucil; cyclosporine,such as, for example, aerosolized cyclosporine (see, U. S. PatentApplication Publication No. US20020006901), tacrolimus (FK506;ProGrafm), mycophenolate mofetil, and azathioprine (6-mercaptopurine),sirolimus (rapamycin), deoxyspergualin, leflunomide and itsmalononitriloamide analogs; immune modulators, including for examplethalidomide and its analogs; and immunosuppressive proteins, including,for example, anti-CTLA antibodies and Ig fusions, anti-B lymphocytestimulator antibodies (e.g., LYMPHOSTAT-BTM) and Ig fusions (BLyS-Ig),anti-CD80 antibodies and etanercept (Enbrel®), as well as anti-T cellantibodies such as anti-CD3 (OKT3), anti-CD4, and the like.

As such, it is specifically contemplated that the compositions andmethods of the invention are used in combination with other drugs tofurther improve symptoms and outcomes in transplant recipients, such asthose receiving lung or kidney grafts, for example. Thus, in someembodiments, the anti-CD40 antibodies or proteins of the invention, forexample mAb1, mAb2 or mAb3 antibody, are used to treat transplantrejection (such as, for example hyperacute, acute, and/or chronicrejection or graft versus host disease in lung or renal transplantrecipients) alone or in combination with parenterally and/ornon-parenterally administered cyclosporine, including for example oralcyclosporine, injectable cyclosporine, aerosolized (e.g.inhaled)cyclosporine, and combinations thereof. In some embodimentswhere at least a component of the therapy is aerosolized cyclosporine,the cyclosporine is delivered to the lung of the recipient by inhalationof cyclosporine in aerosol spray form using, for example, a pressurizeddelivery device or nebulizer. The cyclosporine may be administered ineither dry powder or wet form. The cyclosporine may be administered as asub therapeutic dose.

In some other embodiments, the anti-CD40 antibodies or proteins of theinvention, for example mAb1, mAb2 or mAb3 antibody, may be used alone orin combination with immunosuppressive drugs to treat and/or preventrheumatoid arthritis. Thus in some embodiments where the anti-CD40antibodies or proteins of the invention; for example mAb1, mAb2 or mAb3antibody, are used to treat rheumatoid arthritis, said antibodies orproteins may be used in combination with suitable immunosuppressivedrugs, including, but not limited to, methotrexate, cyclophosphamide,mizoribine, chlorambucil, cyclosporine, tacrolimus (FK506; PROGRAFTM),mycophenolate mofetil, and azathioprine (6-mercaptopurine), sirolimus(rapamycin), deoxyspergualin, leflunomide and its malononitriloamideanalogs; and immunosuppressive proteins, including, for example,anti-CTLA antibodies and Ig fusions, anti-B lymphocyte stimulatorantibodies (e.g., LYMPHOSTAT-BTM) and Ig fusions (BLyS-Ig), anti-CD20antibodies (e.g. RITUXAN (g)); the fully human antibody HuMax-CD20,R-1594, IMMU-106, TRU-015, AME-133, tositumomab/1-131, tositumomab(Bexxar (D), ibritumomab tituxetan (Zcvalin®); anti-CD80 antibodies, andetanercept (ENBREL), as well as anti-T cell antibodies such as anti-CD3(OKT3), anti-CD4, and the like. As discussed above, treatmenteffectiveness may be assessed using any means and includes, but is notlimited to, effectiveness as measured by clinical responses defined bythe American College of Rheumatology criteria, the European League ofRheumatism criteria, or any other criteria. See for example, Felson etal. (1995) Arthritis. Rheum. 38: 727-35 and van Gestel et al. (1996)Arthritis Rheum. 39: 34-40.

In yet other embodiments, the anti-CD40 antibodies or proteins of theinvention, for example mAb1, mAb2 or mAb3 antibody, may be used alone orin combination with immunosuppressive drugs to treat and/or preventmultiple sclerosis. Thus in some embodiments where the anti-CD40antibodies or proteins of the invention, for example mAb1, mAb2 or mAb3antibody, are used to treat multiple sclerosis, the antibodies may usedin combination with suitable immunosuppressive drugs, including, but notlimited to, methotrexate, cyclophosphamide, mizoribine, chlorambucil,cyclosporine, tacrolimus (FK506; PROGRAFTM), mycophenolate mofetil, andazathioprine (6-mercaptopurine), sirolimus (rapamycin), deoxyspergualin,leflunomide and its malononitriloamide analogs; and immunosuppressiveproteins, including, for example, anti-CTLA antibodies and Ig fusions,anti-B lymphocyte stimulator antibodies (e.g., LYMPHOSTAT-BTM) and Igfusions (BLyS-Ig), anti-CD20 antibodies (e.g., RITUXAN (D); the fullyhuman antibody HuMax-CD20, R-1594, IMMIJ-106, TRU-015, AME-133,tositumomab/1-131, tositumomab (Bexxar®), ibritumomab tituxetan(Zevalin®); anti-CD80 antibodies, and etanercept (ENBREL), as well asanti-T cell antibodies such as anti-CD3 (OKT3), anti-CD4, agentsinvolved in S1P receptor modulation, including for example fingolimod;and the like.

Pharmaceutical Formulations and Modes of Administration

The anti-CD40 antibodies or proteins of this invention are administeredat a concentration that is therapeutically effective to prevent or treatautoimmune diseases and/or inflammatory diseases and/or to prevent orreduce risks associated to graft rejection in transplantation.

To accomplish this goal, the antibodies may be formulated using avariety of acceptable excipients known in the art. Typically, theantibodies or proteins are administered by injection, for example,either intravenously, intraperitoneally, or subcutaneously. Methods toaccomplish this administration are known to those of ordinary skill inthe art. It may also be possible to obtain compositions that may betopically or orally administered, or which may be capable oftransmission across mucous membranes.

Intravenous administration occurs preferably by infusion over a periodof about 1 to about 10 hours, more preferably over about 1 to about 8hours, even more preferably over about 2 to about 7 hours, still morepreferably over about 4 to about 6 hours, depending upon the anti-CD40antibody or protein being administered. The initial infusion with thepharmaceutical composition may be given over a period of about 4 toabout 6 hours with subsequent infusions delivered more quickly.Subsequent infusions may be administered over a period of about 1 toabout 6 hours, including, for example, about 1 to about 4 hours, about 1to about 3 hours, or about 1 to about 2 hours.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples ofpossible routes of administration include parenteral, (e.g., intravenous(IV), intramuscular (IM), intradermal, subcutaneous (SC), or infusion),oral and pulmonary (e.g., inhalation), nasal, transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerin, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

The anti-CD40 antibodies or proteins of the invention are typicallyprovided by standard technique within a pharmaceutically acceptablebuffer, for example, sterile saline, sterile buffered water, propyleneglycol, combinations of the foregoing, etc. Methods for preparingparenterally administrable agents are described in Remington'sPharmaceutical Sciences (18th ed.; Mack Publishing Company, Eaton, Pa.,1990). See also, for example, WO 98/56418, which describes stabilizedantibody pharmaceutical formulations suitable for use in the methods ofthe present invention.

The amount of at least one antagonist anti-CD40 antibody or proteins ofthe invention to be administered is readily determined by one ofordinary skill in the art. Factors influencing the mode ofadministration and the respective amount of at least one antagonistanti-CD40 antibody or protein include, but are not limited to, theparticular disease undergoing therapy, the severity of the disease, thehistory of the disease, and the age, height, weight, health, andphysical condition of the individual undergoing therapy. Similarly, theamount of antagonist anti-CD40 antibody or protein to be administeredwill be dependent upon the mode of administration and whether thesubject will undergo a single dose or multiple doses of this agent.Generally, a higher dosage of anti-CD40 antibody or protein is preferredwith increasing weight of the patient undergoing therapy. The dose ofanti-CD40 antibody or protein to be administered is in the range fromabout 0.003 mg/kg to about 50 mg/kg, preferably in the range of 0.01mg/kg to about 40 mg/kg.

Thus, for example, the dose can be 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg,0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 5mg/kg, 7 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35mg/kg, 40 mg/kg, 45 mg/kg, or 50 mg/kg.

In another embodiment of the invention, the method comprisesadministration of multiple doses of antagonist anti-CD40 antibody orfragment thereof. The method may comprise administration of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or more therapeuticallyeffective doses of a pharmaceutical composition comprising an antagonistanti-CD40 antibody or fragment thereof. The frequency and duration ofadministration of multiple doses of the pharmaceutical compositionscomprising anti-CD40 antibody or protein can be readily determined byone of skill in the art. Moreover, treatment of a subject with atherapeutically effective amount of an antibody or protein can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antagonist anti-CD40antibody or protein of the invention in the range of between about 0.1to 20 mg/kg body weight, once per week for between about 1 to 10 weeks,preferably between about 2 to 8 weeks, more preferably between about 3to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.Treatment may occur annually to prevent relapse or upon indication ofrelapse. It will also be appreciated that the effective dosage ofantibody or antigen-binding fragment thereof used for treatment mayincrease or decrease over the course of a particular treatment. Changesin dosage may result and become apparent from the results of diagnosticassays as described herein.

Thus, in one embodiment, the dosing regimen includes a firstadministration of a therapeutically effective dose of at least oneanti-CD40 antibody or protein of the invention on days 1, 7, 14, and 21of a treatment period. In another embodiment, the dosing regimenincludes a first administration of a therapeutically effective dose ofat least one anti-CD40 antibody or protein of the invention on days 1,2, 3, 4, 5, 6, and 7 of a week in a treatment period. Furtherembodiments include a dosing regimen having a first administration of atherapeutically effective dose of at least one anti-CD40 antibody orprotein of the invention on days 1, 3, 5, and 7 of a week in a treatmentperiod; a dosing regimen including a first administration of atherapeutically effective dose of at least one anti-CD40 antibody orprotein of the invention on days 1 and 3 of a week in a treatmentperiod; and a preferred dosing regimen including a first administrationof a therapeutically effective dose of at least one anti-CD40 antibodyor protein of the invention on day 1 of a week in a treatment period.The treatment period may comprise 1 week, 2 weeks, 3 weeks, a month, 3months, 6 months, or a year. Treatment periods may be subsequent orseparated from each other by a day, a week, 2 weeks, a month, 3 months,6 months, or a year. ranges from 0.003 mg/kg to 50 mg/kg, from 0.01mg/kg to 40 mg/kg, from 0.01 mg/kg to 30 mg/kg, from 0.1 mg/kg to 30mg/kg, from 0.5 mg/kg to 30 mg/kg, from 1 mg/kg to 30 mg/kg, from 3mg/kg to 30 mg/kg, from 3 mg/kg to 25 mg/kg, from 3 mg/kg to 20 mg/kg,from 5 mg/kg to 15 mg/kg, or from 7 mg/kg to 12 mg/kg. Thus, forexample, the dose of any one antagonist anti-CD40 antibody orantigen-binding fragment thereof, for example the anti-CD40 monoclonalantibody or protein of the invention, can be 0.003 mg/kg, 0.01 mg/kg,0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2mg/kg, 2.5 mg/kg, 3 mg/kg, 5 mg/kg, 7 mg/kg, 10 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, orother such doses falling within the range of 0.003 mg/kg to 50 mg/kg.The same therapeutically effective dose of an anti-CD40 antibody orprotein of the invention can be administered throughout each week ofantibody dosing.

Alternatively, different therapeutically effective doses of anantagonist anti-CD40 antibody or protein of the invention can be usedover the course of a treatment period.

In some embodiments, the initial therapeutically effective dose of anantagonist anti-CD40 antibody or antigen-binding fragment thereof asdefined elsewhere herein can be in the lower dosing range (i.e., 0.003mg/kg to 20 mg/kg) with subsequent doses falling within the higherdosing range (i.e., from 20 mg/kg to 50 mg/kg).

In alternative embodiments, the initial therapeutically effective doseof an antagonist anti-CD40 antibody or antigen-binding fragment thereofas defined elsewhere herein can be in the upper dosing range (i.e., 20mg/kg to 50 mg/kg) with subsequent doses falling within the lower dosingrange (i.e., 0.003 mg/kg to 20 mg/kg). Thus, in one embodiment, theinitial therapeutically effective dose of the antagonist anti-CD40antibody or antigen-binding fragment thereof is 20 mg/kg to 35 mg/kg,including about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, and about 35mg/kg, and subsequent therapeutically effective doses of the antagonistanti-CD40 antibody or protein of the invention are about 5 mg/kg toabout 15 mg/kg, including about 5 mg/kg, 8 mg/kg, 10 mg/kg, 12 mg/kg,and about 15 mg/kg.

In some embodiments of the invention, anti-CD40 therapy is initiated byadministering a “loading dose” of the antibody or protein of theinvention to the subject in need of anti-CD40 therapy. By “loading dose”is intended an initial dose of the anti-CD40 antibody or protein of theinvention that is administered to the subject, where the dose of theantibody or protein of the invention administered falls within thehigher dosing range (i.e., from about 20 mg/kg to about 50 mg/kg). The“loading dose” can be administered as a single administration, forexample, a single infusion where the antibody or antigen-bindingfragment thereof is administered IV, or as multiple administrations, forexample, multiple infusions where the antibody or antigen-bindingfragment thereof is administered IV, so long as the complete “loadingdose” is administered within about a 24-hour period. Followingadministration of the “loading dose”, the subject is then administeredone or more additional therapeutically effective doses of the anti-CD40antibody or protein of the invention. Subsequent therapeuticallyeffective doses can be administered, for example, according to a weeklydosing schedule, or once every two weeks, once every three weeks, oronce every four weeks. In such embodiments, the subsequenttherapeutically effective doses generally fall within the lower dosingrange (i.e. 0.003 mg/kg to 20 mg/kg).

Alternatively, in some embodiments, following the “loading dose”, thesubsequent therapeutically effective doses of the anti-CD40 antibody orprotein of the invention are administered according to a “maintenanceschedule”, wherein the therapeutically effective dose of the antibody orprotein of the invention is administered once a month, once every 6weeks, once every two months, once every 10 weeks, once every threemonths, once every 14 weeks, once every four months, once every 18weeks, once every five months, once every 22 weeks, once every sixmonths, once every 7 months, once every 8 months, once every 9 months,once every 10 months, once every 11 months, or once every 12 months. Insuch embodiments, the therapeutically effective doses of the anti-CD40antibody or protein of the invention fall within the lower dosing range(i.e., 0.003 mg/kg to about 20 mg/kg), particularly when the subsequentdoses are administered at more frequent intervals, for example, onceevery two weeks to once every month, or within the higher dosing range(i.e., from 20 mg/kg to 50 mg/kg), particularly when the subsequentdoses are administered at less frequent intervals, for example, wheresubsequent doses are administered one month to 12 months apart.

Any pharmaceutical composition comprising an anti-CD40 antibody orprotein of the invention having the desired functional propertiesdescribed herein as the therapeutically active component can be used inthe methods of the invention. Thus liquid, lyophilized, or spray-driedcompositions comprising one or more of the anti-CD40 antibodies orproteins of the invention, for example, mAb1, mAb2 or mAb3 antibodies,may be prepared as an aqueous or non-aqueous solution or suspension forsubsequent administration to a subject in accordance with the methods ofthe invention.

Each of these compositions will comprise at least one of the anti-CD40antibodies or proteins of the present invention as a therapeutically orprophylactically active component.

By “therapeutically or prophylactically active component” is intendedthe anti-CD40 antibody or protein of the invention is specificallyincorporated into the composition to bring about a desired therapeuticor prophylactic response with regard to treatment, prevention, ordiagnosis of a disease or condition within a subject when thepharmaceutical composition is administered to that subject. Preferablythe pharmaceutical compositions comprise appropriate stabilizing agents,bulking agents, or both to minimize problems associated with loss ofprotein stability and biological activity during preparation andstorage.

Formulants may be added to pharmaceutical compositions comprising ananti-CD40 antibody or protein of the invention. These formulant mayinclude, but are not limited to, oils, polymers, vitamins,carbohydrates, amine acids, salts, buffers, albumin, surfactants, orbulking agents. Preferably carbohydrates include sugar or sugar alcoholssuch as mono-, di-, or polysaccharides, or water soluble glucans. Thesaccharides or glucans can include fructose, glucose, mannose, sorbose,xylose, maltose, sucrose, dextran, pullulan, dextrin, α and βcyclodextrin, soluble starch, hydroxyethyl starch, andcarboxymethylcellulose, or mixtures thereof.

“Sugar alcohol” is defined as a C₄ to C₈ hydrocarbon having a hydroxylgroup and includes galactitol, inositol, mannitol, xylitol, sorbitol,glycerol, and arabitol. These sugars or sugar alcohols may be usedindividually or in combination. The sugar or sugar alcohol concentrationmay be between 1.0% and 7% w/v, more preferably between 2.0% and 6.0%w/v. For example, amino acids include levorotary (L) forms of carnitine,arginine, and betaine; however, other amino acids may be added.Preferred polymers include polyvinylpyrrolidone (PVP) with an averagemolecular weight between 2,000 and 3,000, or polyethylene glycol (PEG)with an average molecular weight between 3,000 and 5,000. Surfactantsthat can be added to the formulation are shown in EP Nos. 270,799 and268,110.

Additionally, antibodies can be chemically modified by covalentconjugation to a polymer to increase their circulating half-life, forexample. Preferred polymers, and methods to attach them to peptides, areshown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546.Preferred polymers are polyoxyethylated polyols and polyethylene glycol(PEG). PEG is soluble in water at room temperature and has the generalformula: R(O—CH2-CH2)nO—R where R can be hydrogen, or a protective groupsuch as an alkyl or alkanol group. Preferably, the protective group hasbetween 1 and 8 carbons, more preferably it is methyl. The symbol n is apositive integer, preferably between 1 and 1,000, more preferablybetween 2 and 500. The PEG has a preferred average molecular weightbetween 1,000 and 40,000, more preferably between 2,000 and 20,000, mostpreferably between 3,000 and 12,000. Preferably, PEG has at least onehydroxy group, more preferably it is a terminal hydroxy group. It isthis hydroxy group which is preferably activated to react with a freeamino group on the inhibitor. However, it will be understood that thetype and amount of the reactive groups may be varied to achieve acovalently conjugated PEG/antibody of the present invention.

Water-soluble polyoxyethylated polyols are also useful in the presentinvention.

They include polyoxyethylated sorbitol, polyoxyethylated glucose,polyoxyethylated glycerol (POG), and the like. POG is preferred. Onereason is because the glycerol backbone of polyoxyethylated glycerol isthe same backbone occurring naturally in, for example, animals andhumans in mono-, di-, triglycerides. Therefore, this branching would notnecessarily be seen as a foreign agent in the body. The POG has apreferred molecular weight in the same range as PEG. The structure forPOG is shown in Knauf et al. (1988, J. Bio. Chem. 263: 15064-15070) anda discussion of POG/IL-2 conjugates is found in U.S. Pat. No. 4,766,106.

Another drug delivery system for increasing circulatory half-life is theliposome.

Methods of preparing liposome delivery systems are discussed in Gabizonet al. (1982) Cancer Research 42: 4734; Cafiso (1981) Biochem BiophysActa 649: 129; and Szoka (1980) Ann. Rev. Bioplays. Eng. 9: 467. Otherdrug delivery systems are known in the art and are described in, e.g.,Poznansky et al. (1980) Drug Delivery Systems (R. L. Juliano, ed.,Oxford, N. Y.) pp. 253-315; Poznansky (1984) Pharm Revs 36: 277.

The formulant to be incorporated into a pharmaceutical compositionshould provide for the stability of the antagonist anti-CD40 antibody orprotein of the invention. That is, the anti-CD40 antibody or protein ofthe invention should retain its physical and/or chemical stability andhave the desired functional properties, i.e., one or more of the desiredfunctional properties defined herein above.

Methods for monitoring protein stability are well known in the art. See,for example, Jones (1993) Adv. Drug Delivery Rev. 10: 29-90; Lee, ed.(1991) Peptide and Protein Drug Delivery (Marcel Dekker, Inc., New York,N.Y.); and the stability assays disclosed herein below. Generally,protein stability is measured at a chosen temperature for a specifiedperiod of time. In preferred embodiments, a stable antibodypharmaceutical formulation provides for stability of the antagonistanti-CD40 antibody or protein of the invention when stored at roomtemperature (about 25° C.) for at least 1 month, at least 3 months, orat least 6 months, and/or is stable at about 2-8 C for at least 6months, at least 9 months, at least 12 months, at least 18 months, atleast 24 months.

A protein such as an antibody, when formulated in a pharmaceuticalcomposition, is considered to retain its physical stability at a givenpoint in time if it shows no visual signs (i.e., discoloration or lossof clarity) or measurable signs (for example, using size-exclusionchromatography (SEC) or UV light scattering) of precipitation,aggregation, and/or denaturation in that pharmaceutical composition.With respect to chemical stability, a protein such as an antibody, whenformulated in a pharmaceutical composition, is considered to retain itschemical stability at a given point in time if measurements of chemicalstability are indicative that the protein (i.e., antibody) retains thebiological activity of interest in that pharmaceutical composition.Methods for monitoring changes in chemical stability are well known inthe art and include, but are not limited to, methods to detectchemically altered forms of the protein such as result from clipping,using, for example, SDS-PAGE, SEC, and/or matrix-assisted laserdesorption ionization/time of flight mass spectrometry; and degradationassociated with changes in molecular charge (for example, associatedwith deamidation), using, for example, ion-exchange chromatography. See,for example, the methods disclosed herein below.

An anti-CD40 antibody or protein of the invention, when formulated in apharmaceutical composition, is considered to retain a desired biologicalactivity at a given point in time if the desired biological activity atthat time is within about 30%, preferably within about 20% of thedesired biological activity exhibited at the time the pharmaceuticalcomposition was prepared as determined in a suitable assay for thedesired biological activity. Assays for measuring the desired biologicalactivity of the antagonist anti-CD40 antibodies or proteins disclosedherein, can be performed as described in the Examples herein. See alsothe assays described in Schutze et al. (1998) Proc. Natl. Acad. Sci. USA92: 8200-8204; Denton et al. (1998) Pediatr Transplant. 2: 6-15; Evanset al. (2000) J: Immunol. 164: 688-697; Noelle (1998) Agents ActionsSuppl. 49: 17-22; Lederman et al. (1996) Curr Opin. Hematol. 3: 77-86;Coligan et al. (1991) CurrentProtocols in Immunology 13: 12; Kwekkeboomet al. (1993) Immunology 79: 439-444; and U.S. Pat. Nos. 5,674,492 and5,847,082.

In some embodiments of the invention, the anti-CD40 antibody, forexample, selected among mAb1-mAb3 recombinant antibodies, is formulatedin a liquid pharmaceutical formulation. The anti-CD40 antibody orprotein of the invention can be prepared using any method known in theart, including those methods disclosed herein above. In one embodiment,the anti-CD40 antibody, for example, selected among the mAb1-mAb3antibodies, is recombinantly produced in a CHO cell line.

Following its preparation and purification, the anti-CD40 antibody canbe formulated as a liquid pharmaceutical formulation in the manner setforth herein. Where the antagonist anti-CD40 antibody is to be storedprior to its formulation, it can be frozen, for example, at −20° C., andthen thawed at room temperature for further formulation.

The liquid pharmaceutical formulation comprises a therapeuticallyeffective amount of the anti-CD40 antibody or protein of the invention,for example, mAb1, mAb2 or mAb3 antibody. The amount of antibody presentin the formulation takes into consideration the route of administrationand desired dose volume.

In this manner, the liquid pharmaceutical composition comprises theanti-CD40 antibody, for example, mAb1, mAb2 or mAb3 antibody, at aconcentration of 0.1 mg/ml to 300.0 mg/ml, 1.0 mg/ml to 200 mg/ml, 5.0mg/ml to 100.0 mg/ml, 7.5 mg/ml to 50 mg/ml, or 15.0 mg/ml to 25.0mg/ml.

The liquid pharmaceutical composition comprises the anti-CD40 antibody,for example, mAb1, mAb2 or mAb3 antibody and a buffer that maintains thepH of the formulation in the range of pH 5.0 to pH 7.0.

Any suitable buffer that maintains the pH of the liquid anti-CD40antibody formulation in the range of about pH 5.0 to about pH 7.0 can beused in the formulation, so long as the physicochemical stability anddesired biological activity of the antibody are retained as noted hereinabove. Suitable buffers include, but are not limited to, conventionalacids and salts thereof, where the counter ion can be, for example,sodium, potassium, ammonium, calcium, or magnesium. Examples ofconventional acids and salts thereof that can be used to buffer thepharmaceutical liquid formulation include, but are not limited to,succinic acid or succinate, histidine or histidine hydrochloride, citricacid or citrate, acetic acid or acetate, tartaric acid or tartarate,phosphoric acid or phosphate, gluconic acid or gluconate, glutamic acidor glutamate, aspartic acid or aspartate, maleic acid or maleate, andmalic acid or malate buffers. The buffer concentration within theformulation can be from 1 mM to 50 mM, including about 1 mM, 2 mM, 5 mM,8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, orother such values within the range of 1 mM to 50 mM.

In some embodiments of the invention, the liquid pharmaceuticalformulation comprises a therapeutically effective amount of theanti-CD40 antibody, for example, the mAb1, mAb2 or mAb3 antibody, andsuccinate buffer or citrate buffer or histidine buffer or histidinehydrochloride buffer at a concentration that maintains the pH of theformulation in the range of about pH 5.0 to pH 7.0. By “succinatebuffer” or “citrate buffer” is intended a buffer comprising a salt ofsuccinic acid or a salt of citric acid, respectively. By “histidinebuffer” is intended a buffer comprising a salt of the amino acidhistidine.

In a preferred embodiment, the buffer is a histidine buffer, e.g.,histidine hydrochloride. As noted above, the histidine bufferconcentration within the formulation can be from 1 mM to 50 mM,including about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30mM, 35 mM, 40 mM, 45 mM, 50 mM, or other such values within the range of1 mM to 50 mM.

In a preferred embodiment, the succinate or citrate counterion is thesodium cation, and thus the buffer is sodium succinate or sodiumcitrate, respectively. However, any cation is expected to be effective.Other possible succinate or citrate cations include, but are not limitedto, potassium, ammonium, calcium, and magnesium. As noted above, thesuccinate or citrate buffer concentration within the formulation can befrom 1 mM to 50 mM, including about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or other suchvalues within the range of 1 mM to 50 mM.

In other embodiments, the liquid pharmaceutical formulation comprisesthe antagonist anti-CD40 antibody, for example, the mAb1, mAb2 or mAb3antibody, at a concentration of 0.1 mg/ml to 300.0 mg/ml, or 1.0 mg/mlto 200 mg/ml, 5.0 mg/ml to 100.0 mg/ml, 7.5 mg/ml to 50 mg/ml, or 15.0mg/ml to 25.0 mg/ml, and histidine or succinate or citrate buffer, forexample, sodium succinate or sodium citrate buffer or histidinehydrochloride, at a concentration of 1 mM to 50 mM, 5 mM to 40 mM, 10 mMto 35 mM, preferably about 30 mM.

Where it is desirable for the liquid pharmaceutical formulation to benear isotonic, the liquid pharmaceutical formulation comprising atherapeutically effective amount of the anti-CD40 antibody or protein ofthe invention, for example, the mAb1, mAb2 or mAb3 antibody, and abuffer to maintain the pH of the formulation within the range of aboutpH 5.0 to about pH 7.0 can further comprise an amount of an isotonizingagent sufficient to render the formulation near isotonic. By “nearisotonic” is intended the aqueous formulation has an osmolarity of 240mmol/kg to 800 mmol/kg, preferably about 240 to about 600 mmol/kg, morepreferably about 240 to about 440 mmol/kg, more preferably about 250 toabout 330 mmol/kg, even more preferably about 260 to about 320 mmol/kg,still more preferably about 270 to about 310 mmol/kg.

Methods of determining the isotonicity of a solution are known to thoseskilled in the art. See, for example, Setnikar et al. (1959) J. Am.Pharm. Assoc. 48:628. Those skilled in the art are familiar with avariety of pharmaceutically acceptable solutes useful in providingisotonicity in pharmaceutical compositions. The isotonizing agent can beany reagent capable of adjusting the osmotic pressure of the liquidpharmaceutical formulation of the present invention to a value nearlyequal to that of a body fluid. It is desirable to use a physiologicallyacceptable isotonizing agent.

Thus, the liquid pharmaceutical formulation comprising a therapeuticallyeffective amount of the antagonist anti-CD40 antibody, for example, themAb1, mAb2 or mAb3 antibody and a buffer to maintain the pH of theformulation within the range of about pH 5.0 to about pH 7.0, canfurther comprise components that can be used to provide isotonicity, forexample, sodium chloride; amino acids such as alanine, valine, andglycine; sugars and sugar alcohols (polyols), including, but not limitedto, glucose, dextrose, fructose, sucrose, maltose, mannitol, trehalose,glycerol, sorbitol, and xylitol; acetic acid, other organic acids ortheir salts, and relatively minor amounts of citrates or phosphates. Theordinary skilled person would know of additional agents that aresuitable for providing optimal tonicity of the liquid formulation.

In some preferred embodiments, the liquid pharmaceutical formulationcomprising a therapeutically effective amount of the anti-CD40 antibody,for example, the mAb1, mAb2 or mAb3 antibody, and a buffer to maintainthe pH of the formulation within the range of about pH 5.0 to about pH7.0, further comprises sodium chloride as the isotonizing agent.

The concentration of sodium chloride in the formulation will depend uponthe contribution of other components to tonicity. In some embodiments,the concentration of sodium chloride is 50 mM to 300 mM. In one suchembodiment, the concentration of sodium chloride is about 150 mM. Inother such embodiments, the concentration of sodium chloride is about150 mM, the buffer is sodium succinate or sodium citrate buffer at aconcentration of 5 mM to 15 mM, the liquid pharmaceutical formulationcomprises a therapeutically effective amount of the anti-CD40 antibodyor protein of the invention, for example, the mAb1, mAb2 or mAb3antibody, and the formulation has a pH of 5.0 to pH 7.0.

In other embodiments, the liquid pharmaceutical formulation comprisesthe anti-CD40 antibody or protein of the invention, for example, mAb1,mAb2 or mAb3 antibody, at a concentration of 0.1 mg/ml to 50.0 mg/ml or5.0 mg/ml to 25.0 mg/ml, about 150 mM sodium chloride, and about 10 mM,20 mM 30 mM, 40 mM or 50 mM sodium succinate or sodium citrate, at a pHof about pH 5.5.

Protein degradation due to freeze thawing or mechanical shearing duringprocessing of a liquid pharmaceutical formulation of the presentinvention can be inhibited by incorporation of surfactants into theformulation in order to lower the surface tension at the solution-airinterface. Thus, in some embodiments, the liquid pharmaceuticalformulation comprises a therapeutically effective amount of theanti-CD40 antibody or protein of the invention, for example, the mAb1,mAb2 or mAb3 antibody, a buffer to maintain the pH of the formulationwithin the range of about pH 5.0 to about pH 7.0, and further comprisesa surfactant. In other embodiments, the liquid pharmaceuticalformulation comprises a therapeutically effective amount of theanti-CD40 antibody or protein of the invention, for example, the mAb1,mAb2 or mAb3 antibody, a buffer to maintain the pH of the formulationwithin the range of about pH 5.0 to about pH 7.0, an isotonizing agentsuch as sodium chloride at a concentration of about 50 mM to about 300mM, and further comprises a surfactant.

Typical surfactants employed are nonionic surfactants, includingpolyoxyethylene sorbitol esters such as polysorbate 80 (Tween 80) andpolysorbate 20 (Tween 20); polyoxypropylene-polyoxyethylene esters suchas Pluronic F68; polyoxyethylene alcohols such as Brij 35; simethicone;polyethylene glycol such as PEG400; lysophosphatidylcholine; andpolyoxyethylene-p-t-octylphenol such as Triton X-100.

Classic stabilization of pharmaceuticals by surfactants or emulsifiersis described, for example, in Levine et al. (1991) J. Parenteral Sci.Technol. 45 (3): 160-165, herein incorporated by reference. A preferredsurfactant employed in the practice of the present invention ispolysorbate 80. Where a surfactant is included, it is typically added inan amount from 0.001% to 1.0% (w/v).

Thus, in some embodiments, the liquid pharmaceutical formulationcomprises a therapeutically effective amount of the anti-CD40 antibodyor protein of the invention, for example, the mAb1, mAb2 or mAb3antibody, the buffer is sodium succinate or sodium citrate or histidinebuffer or histidine hydrochloride buffer at a concentration of 1 mM to50 mM, 3 mM to 40 mM, or 5 mM to 35 mM; or 7.5 mM to 30 mM; theformulation has a pH of pH 5.0 to pH 7.0; and the formulation furthercomprises a surfactant, for example, polysorbate 80, in an amount from0.001% to 1.0% or 0.001% to 0.5%. Such formulations can optionallycomprise an isotonizing agent, such as sodium chloride at aconcentration of 50 mM to 300 mM, 50 mM to 200 mM, or 50 mM to 150 mM.

In other embodiments, the liquid pharmaceutical formulation comprisesthe anti-CD40 antibody or protein of the invention, for example, themAb1, mAb2 or mAb3 antibody, at a concentration of 0.1 mg/ml to 200.0mg/ml or 1 mg/ml to 100.0 mg/ml or 2 mg/ml to 50.0 mg/ml or 5.0 mg/ml to25.0 mg/ml, including about 20.0 mg/ml; 50 mM to 200 mM sodium chloride,including about 150 mM sodium chloride; sodium succinate or sodiumcitrate at 5 mM to 20 mM, including about 10 mM sodium succinate orsodium citrate; sodium chloride at a concentration of 50 mM to 200 mM,including about 150 mM; histidine or histidine chloride at 5 mM to 50mM, including about 30 mM histidine or histidine chloride; andoptionally a surfactant, for example, polysorbate 80, in an amount from0.001% to 1.0%, including 0.001% to 0.5%; where the liquidpharmaceutical formulation has a pH of about pH 5.0 to about pH 7.0.

The liquid pharmaceutical formulation can be essentially free of anypreservatives and other carriers, excipients, or stabilizers notedherein above. Alternatively, the formulation can include one or morepreservatives, for example, antibacterial agents, pharmaceuticallyacceptable carriers, excipients, or stabilizers described herein aboveprovided they do not adversely affect the physicochemical stability ofthe antagonist anti-CD40 antibody or antigen-binding fragment thereof.Examples of acceptable carriers, excipients, and stabilizers include,but are not limited to, additional buffering agents, co-solvents,surfactants, antioxidants including ascorbic acid and methionine,chelating agents such as EDTA, metal complexes (for example, Zn-proteincomplexes), and biodegradable polymers such as polyesters. A thoroughdiscussion of formulation and selection of pharmaceutically acceptablecarriers, stabilizers, and isomolytes can be found in Remington'sPharmaceutical Sciences (18th ed.; Mack Publishing Company, Eaton, Pa.,1990).

After the liquid pharmaceutical formulation or other pharmaceuticalcomposition described herein is prepared, it can be lyophilized toprevent degradation. Methods for lyophilizing liquid compositions areknown to those of ordinary skill in the art. Just prior to use, thecomposition may be reconstituted with a sterile diluent (Ringerssolution, distilled water, or sterile saline, for example) that mayinclude additional ingredients.

Upon reconstitution, the composition is preferably administered tosubjects using those methods that are known to those skilled in the art.

Use of Antagonist Anti-CD40 Antibodies in the Manufacture of Medicaments

The present invention also provides an antagonist anti-CD40 antibody orproteins of the invention for use in treating an autoimmune diseaseand/or inflammatory disease in a subject, wherein the medicament iscoordinated with treatment with at least one other therapy.

By “coordinated” is intended the medicament is to be used either priorto, during, or after treatment of the subject with at least one othertherapy. Examples of other therapies include, but are not limited to,those described herein above, i.e., surgery or surgical procedures (e.g.splenectomy, lymphadenectomy, thyroidectomy, plasmaphoresis,leukophoresis, cell, tissue, or organ transplantation, organ perfusion,intestinal procedures, and the like), radiation therapy, therapy such assteroid therapy and non-steroid therapy, hormone therapy, cytokinetherapy, therapy with dermatological agents (for example, topical agentsused to treat skin conditions such as allergies, contact dermatitis, andpsoriasis), immunosuppressive therapy, and other anti-inflammatorymonoclonal antibody therapy, and the like, where treatment with theadditional therapy, or additional therapies, occurs prior to, during, orsubsequent to treatment of the subject with the medicament comprisingthe antagonist anti-CD40 antibody or proteins of the invention as notedherein above.

In one such embodiment, the present invention provides for mAb1, mAb2 ormAb3 antibody for use in the treatment of an autoimmune disease and/orinflammatory disease in a subject, wherein the medicament is coordinatedwith treatment with at least one other therapy as noted herein above.

In some embodiments, the medicament comprising the antagonist anti-CD40antibody, for example, the monoclonal antibody mAb1, mAb2 or mAb3disclosed herein, or antigen-binding fragment thereof is coordinatedwith treatment with two other therapies.

Where the medicament comprising the antagonist anti-CD40 antibody iscoordinated with two other therapies, use of the medicament can be priorto, during, or after treatment of the subject with either or both of theother therapies.

The invention also provides for an antagonist anti-CD40 antibody, forexample, the antibody mAb1, mAb2 or mAb3 disclosed herein, for use intreating an autoimmune disease and/or inflammatory disease in a subject,wherein the medicament is used in a subject that has been pretreatedwith at least one other therapy.

By “pretreated” or “pretreatment” is intended the subject has beentreated with one or more other therapies prior to receiving themedicament comprising the antagonist anti-CD40 antibody or protein ofthe invention.

The following examples are offered by way of illustration and not by wayof limitation.

FIGURE LEGENDS

FIG. 1 shows incorporation of ³H-thymidine after 72 hours of human PBMCculture stimulated with a dose response of Chir12.12 (empty circle),mAb1 (filled circle), mAb2 (filled square), mAb3 (filled triangle) orhuman CD40L (empty square).

FIG. 2 shows incorporation of ³H-thymidine after 72 hours of human PBMCculture stimulated with a dose response of Chir12.12 (empty circle),mAb1 (filled circle), mAb2 (filled square), mAb3 (filled triangle),human CD40L (empty square), isotype control (filled diamond)co-stimulated in the presence of either 5 μg/ml anti-IgM F(ab′)₂ or 1 μMCpG2006.

FIG. 3 shows incorporation of ³H-thymidine after 72 hours of human PBMCculture stimulated with a dose response of Chir12.12 (empty circle),mAb1 (filled circle), mAb2 (filled square), mAb3 (black triangle), humanCD40L (empty square), isotype control (filled diamond) co-stimulated inthe presence of 75 ng/ml IL-4.

FIG. 4 shows incorporation of ³H-thymidine after 72 hours of human PBMCculture stimulated with 20 μg/ml CD40L with a dose response of Chir12.12(empty circle), mAb1 (filled circle), mAb2 (filled square), mAb3 (blacktriangle) or human CD40L (empty square).

FIG. 5 shows dose-dependent binding of anti-human CD40 antibody to BJABcell lines, respectively, Chir12.12 (filled circle), mAb1 (emptysquare), mAb2 (empty triangle), mAb3 (filled triangle).

EXAMPLES Materials 1. Monoclonal Antibodies

Chir12.12 (1.9 mg/ml), mAb1 (0.88 mg/ml), mAb2 (1.9 mg/ml), and mAb3(1.9 mg/ml) were provided in 50 mM Citrate pH 7.0, 140 mM NaCl. An IgGisotype control was also used for select experiments (Sigma, St. Louis,USA).

2. B Cell Activation Stimuli

AfiniPure F(ab′)2 fragment rabbit anti-human IgM was obtained fromJackson Immuno Research (Suffolk, UK), and CpG2006 was obtained fromMicrosynth (Balgach, Switzerland). Recombinant human CD40L was generatedusing standard procedures known to those of ordinary skill in the art.Supernatant containing human IL-4 was generated using standardprocedures known to those of ordinary skill in the art.

3. In Vitro Tissue Culture Reagents

PBMC culture media: RPMI-1640, 10% FBS, 1% Penicillin/Streptomycin, 1%non essential amino acids, 1% Sodium pyruvate, 5 mM β-mercaptoethanol(all from Invitrogen, San Diego, USA).

Methods 1. CD40L-Mediated PBMC Proliferation Assay 1.1 Purification ofHuman Peripheral Blood Mononuclear Cells (PBMCs)

Primary PBMCs were purified from whole blood buffy coats obtained fromhealthy volunteers (Blutspendezentrum, Basel). Buffy coats were diluted1:4 with Ca2+ and Mg2+ free PBS containing 5 mM EDTA and 25 ml wasaliquoted into 50 ml Falcon tubes. Diluted buffy coats were underlayedwith 14 ml of Ficoll-Plaque Plus (GE Healthcare) per Falcon tube andcentrifuged at room temperature for 20 min at 2250 rpm (no brake).Following centrifugation, the interphase layer was transferred to asingle 50 ml Falcon tube. Interphase layers from multiple tubes (from asingle donor) were combined up to a volume of 30 ml. PBS supplementedwith 5 mM EDTA was added and cells were spun at room temperature for 5min at 2250 rpm. The supernatant was discarded prior to addition of 15ml red blood cell (RBC) lysis buffer and incubation at room temperaturefor 5 min. Subsequently 20 ml of PBS/5 mM EDTA was added and cells werespun again (RT/5 min at 2250 rpm). Cells were washed twice in PBS/5 mMEDTA (with intervening centrifugation steps) and re-suspended in 35 mlPBMC media prior to viable cell number determination using Trypan Bluedye exclusion. Cells not used immediately for in vitro stimulation werecryopreserved.

1.2 In Vitro PBMC Stimulation Assay

Seven point two-fold dilution series of each anti-CD40 or isotypecontrol mAb were made in triplicate in Costar 96 well plates in thepresence or absence of a constant dose of 5 μg/ml anti-IgM F(ab′)2, 1 μMCpG2006, supernatant containing human IL-4 (75 ng/ml), or 40 μg/mlrecombinant huCD40L (final concentrations indicated). Startingconcentrations of each anti-CD40 mAb ranged from 20 μg/ml to 100 μg/mldepending on the experiment. CD40L was used in dose response as apositive control for all experiments. Doses of anti-IgM, CpG2006, IL-4and CD40L were selected based on prior experiments where the ability ofthese reagents (alone or in combination) to induce PBMC or B cellproliferation was assessed in dose response (data not shown). PBMCs(final density of 8×10⁴ per well) were subsequently added to each wellprior to incubation for 3 days at 37° C./5% CO₂. ³H-thymidine (1μCi/50μl/well) was added to each well for the final 6 hours of cultureprior to harvesting and determination of thymidine incorporation using aMicroBetaTrilux scintillation counter. Note that cells plus media andcells plus media plus anti-IgM, IL-4, CpG2006 or CD40L control cultures(in the absence of anti-CD40 mAbs) were included in each experiment.

Scintillation data was analyzed using Excel and GraphPad Prism software.Results are presented as mean counts per minute (cpm) (+/−standard errorof the mean) versus a log transformation of the anti-CD40 mAbconcentration. Positive controls and cell plus media background levelsare indicated on each graph. IC50 or EC50 values were calculated subjectto successful curve-fitting of data by Prism.

PBMCs were stimulated as indicated above with 20 μg/ml CD40L in thepresence or absence of a dose response of the test anti-CD40 mAb for 3days. Proliferation was assessed by ³H-thymidine incorporation after 72hours of culture. Results are presented as the mean of triplicatecultures with SEM and are representative of 4 donors (independentexperiments). IC50 values for anti-CD40 mAb mediated inhibition aretabulated in μg/ml.

2. In Vitro PBMC Agonist Assay

Human PBMCs were stimulated as indicated in paragraph 1.2 above with adose response of Chir12.12, mAb1, mAb2 or mAb3 for 3 days either in theabsence of co-stimulation or in the presence of either 5 μg/ml anti-IgMF(ab′)2 or 1 μM CpG2006. Proliferation was assessed by ³H-thymidineincorporation after 72 hours of culture. Results are presented as themean of triplicate cultures with SEM and are representative of 4 donors(independent experiments).

3. ADCC Assay

50 μl of a PBMC suspension (10×10⁶ cells/mL) was added to round-bottomwells (Corning Incorporated—Costar #3790), 50 μl calcein-stained Rajicells at (2×105 cells/mL) and 100 μl of antibody dilution or controlswere added. Maximum lysis was determined in 2% Triton 100.

Cells were collected at the bottom of the plate (3 minutes at 250 g) andincubated at 37° C. in humidified CO₂ atmosphere (5%) for 1 hour. Cellswere separated from the medium by centrifugation (3 minutes at 750 g)and 100 μl of supernatant were transferred into a clear bottom blackplate (Corning Incorporated—Costar #3904) for measurement.

Fluorescence was determined at 535 nm after excitation at 485 nm with aSpectraMax Gemini spectrometer (Molecular Devices). Specific lysis wascalculated using the following formula:

(experimental release−spontaneous release)/(maximum release−spontaneousrelease)×100.

4. Binding of Anti-Human CD40 Antibody Variants to Human BJAB Cell Line

Flow cytometry was used, in order to compare the binding of thedifferent anti-CD40 antibody Fc variants in their binding to human BJABcells. Therefore, 2×10⁵ cells were seeded per well in a 96-well V-bottomplate. The plates were washed twice with 200 μL of FACS buffer (PBS, 5%FCS, 2 mM EDTA) for 2 min at 4° C. at 1350×g. Supernatants werediscarded and cells were resuspended in 100 mL FACS buffer containing 8%human serum (InVitromex, Cat. No. S4190) and incubated for 10 min. Aftertwo washes anti-human CD40 Fc variants were added in 50 μL FACS bufferwith 1% human serum starting at a concentration of 10 μg/mL in a 1:2dilution. Cells were incubated for 30 min on ice followed by two washingsteps. 50 μL of polyclonal rabbit anti-human IgG FITC, F(ab′)2 (DAKO,Cat. No. F 0315) were added to each well and incubated for 30 min onice. At the end of the incubation cells were washed twice, resuspendedin 100 μL FACS buffer and acquired on a FACS Cantoll.

After acquisition the mean fluorescence intensity of the FITC channelwas acquired in FloJo.Graphing and curve fitting was performed withGraphPad Prism 5.0. Due to changing intensities between individualexperiments, values were normalized. Therefore, the highest value ofeach antibody test series was equaled 100%. Non-linear curve fittingswere performed with the percent values.

5. CD40L-Mediated Cytokine Production Assay in Monocyte-DerivedDendritic Cells (MoDCs) 5.1 Preparation of Human Monocyte-DerivedDendritic Cells

Human PBMCs were prepared from human buffy coats provided by the SwissRed Cross. The buffy coat was diluted 1:5 in PBS (Invitrogen, Cat. No.20012019) and distributed in 35 mL aliquots to 50 mL falcon tubes.Subsequently, 13 mL of Ficoll (GE Healthcare, Cat. No. 17 1440-02) wereunderlain in each tube. Cells were centrifuged at 1680×g at RT for 20min without break. The PBMC containing layer was collected and washedtwice in a large volume of PBS for 5 min at 1000×g. Finally cells wereresuspended in 10 mL PBS and counted.

Human monocytes were negatively isolated from the 100×10⁷ PBMCs usingthe human monocyte isolation kit II from Miltenyi (Miltenyi, Germany,Cat. No. 130-091-153) on an AutoMACS instrument according to themanufacturer's instructions. After the isolation cells were washed twicefor 5 min at 1000×g at 4° C. in culture medium (RPMI1640 (Invitrogen,Cat. No. 61870010), 10% FCS (Invitrogen, Cat. No. 16000044, US origin),1 mM sodium pyruvate (Invitrogen, Cat. No. 11360039), 1×NEAA(Invitrogen, Cat. No. 11140035) 1×Penicillin/Streptomycin (Invitrogen,Cat. No. 15140122)). Isolated monocytes were counted, plated in 6 wellplates at a density of 0.4×106/mL and cultured for seven days at 37° C.,5% CO2. To differentiate the monocytes to dendritic cells recombinant,human IL-4 [80 ng/mL] and human GM-CSF [100 ng/mL] (both produced inhouse) were added to the culture medium at the start of the culture.

5.2 Stimulation of Dendritic Cells (DCs) for Cytokine Release

Immature DCs were harvested after 7 days of culture by rinsing the 6well plates, pooling the cells and washing them twice for 5 min at1400×g in culture medium. Subsequently, 2×105 iDCs were seeded in96-well flat-bottom plates (Becton Dickinson, Cat. No. 353072) in 100μL. For the positive control cells were stimulated with MegaCD40L(Alexis, Cat. No. ALX-522-110-0010) at a concentration of 1 μg/mL, thenegative control consisted of iDCs in medium only. In the antagonismassay anti-human CD40 antibody Fc variants were added at 10 μg/mL in a1:2 dilution together with 1 μg/mL MegaCD40L for a dose-response.Supernatants of the stimulated cells were collected 24 h later for themeasurement of TNFα. In the agonism assay anti-human CD40 antibody Fcvariants were added at 10 μg/mL in a 1:2 dilution only and supernatantswere collected after 48 h for the measurement of TNFα. Cells were seededand stimulated in triplicates for all assays.

5.3 Measurement of TNF Alpha by ELISA

To measure the amount of TNFα in the supernatants, ELISA was performedas follows. Anti-TNFα capture antibody (BD Pharmingen, Cat. No. 551220)was coated on ELISA plates (Greiner, Nunc F96 Maxisorp, Cat. No. 442404)at 5 μg/mL in 50 μL per well overnight at 4° C. For every washing stepplates were washed 3× with 250 μL in a BioTek ELx 405 plate washer.After the first wash, 200 μL of Superblock TBS (Thermo Scientific,Pierce, Cat. No. 37535) were incubated for 1 h at 37° C. Next, TNFαstandard (recombinant human TNFα, R&D Systems, Cat. No. 210-TA) orsample were added in 25 μl. The standard started at a finalconcentration of 20 ng/mL in a 1:2 dilution series. In addition, 25 μLof detection antibody (anti-human TNFα biotin, BD Pharmingen, Cat. No.554511) was added in a 1:500 dilution. Plates were incubated overnightat 4° C. After washing, Avidin-POD conjugate (ExtrAV alkalinephosphatase, Sigma, Cat. No. E-2636) was diluted 1:5000 in SuperblockTBS, added in 50 μL and incubated for 1 h at RT. Plates were washed and50 μL of the substrate p-Nitrophenylphosphat (Sigma, Cat. No. C-3041)was added to develop for 15 min. ELISA plates were read at 450 nm withthe software SoftMax Pro on a SpectraMax M5 (Molecular Devices).

6. Toxicology Study

The primary initial purpose of the toxicology study was to investigatethe potential toxicity of high dose (100 mg/kg) mAb1 in comparison toChir12.12.

Thirty cynomolgus monkeys of Mauritian origin were used for this study.At the initiation of dosing, the animals were approximately 4 to 5 yearsof age and weighed 4.5 to 6.6 kg for the males and 3.1 to 4.3 kg for thefemales.

mAb1 (50 mg/mL) was administered intravenously to one group ofcynomolgus monkeys (5 males/5 females; group 2) at a dose level of 100mg/kg and a dose volume of 2 mL/kg once weekly for 5 weeks (test itemapplications on days 1, 8, 15, 22, 29, and 36). A further group ofcynomolgus monkeys (5 males/5 females; group 3) received the parentantibody Chir12.12 intravenously by slow bolus infusion at a dose levelof 100 mg/kg and a dose volume of 5 mL/kg. mAb1 placebo at a dose volumeof 2 mL/kg was given to another group of cynomolgus monkeys which servedas controls (5 males/5 females; group 1). All animals were subjected tonecropsy one or two days after the last dosing.

It was decided to incorporate Keyhole Limpet Hemocyanin(KLH)-immunization in order to evaluate the efficacy of both anti-CD40Abs. On day 2 animals were immunized with 1 mg KLH in Alum followed by abooster injection of 0.5 mg KLH in Alum on day 23. Serum was sampledpre-immunization/booster as well as on days 7 and 14 after immunizationand booster, respectively. KLH specific IgM/IgG titers were determinedwith ELISA using cynomolgus monkey anti KLH IgM/IgG reference serum asstandard. Blood was sampled on 3 pre-dose occasions and on day 15, 29and at necropsy (day 37/38) for immunophenotyping of naive B cells(CD20+CD21+CD27−). Absolute naive CD20 B cell counts were calculatedfrom the total lymphocyte count per blood sample and the relative naiveCD20 B cell count by flow cytometry.

TABLE 4 Summary of toxicology study Dose Group Group Dose level volume*Animals/group Necropsy after number description (mg/kg/dose) (mL/kg)Male Female 5 weeks 1 Control 0 2 5 5 5 M/5 F 2 mAb1 100 2 5 5 5 M/5 F 3Chir12.12 100 5 5 5 5 M/5 F *Based on most recent individual body weight

Assessment of toxicity was based on mortality, clinical observations(clinical signs including post-dosing observations, feces evaluation,fur inspection, and food consumption), body weights, ophthalmicexaminations, cardiovascular investigations, clinical pathology(including coagulation, external platelet activation examinations,hematology, clinical chemistry and urine analysis), organ weights, andmacroscopic and microscopic necropsy findings. Blood immunophenotypingwas performed three times predose, on days 15 and 29 of the dosing phaseas well as on the day of necropsy. Furthermore, immunophenotyping ofspleen tissue and draining lymph nodes of the Keyhole Limpet Hemocyanin(KLH) injection site was performed at necropsy. In addition, the T-celldependent antibody response (TDAR) to KLH was examined to directlycompare the influence of the fully ADCC-capable Chir12.12 with the mAb1.Blood was collected from all animals for toxicokinetic evaluation andfor a possible anti-drug-antibody (ADA) evaluation.

7. Additional In Vitro Profiling of mAb1

7.1 PBMC Purification

Human peripheral blood mononuclear cells were prepared as describedpreviously in section 1.1.

7.2 Human Tonsil B Cell Purification

The tonsil capsule and connective tissue was removed and tonsil materialafter was cut the tonsil into ˜5 mm big pieces prior to being mashedthrough a metal cell strainer with regular washing with B cell media.Tonsilar cells were then filtered twice through a 70 μM cell strainer inorder to remove cellular debris. B cells were isolated from fresh PBMCsusing an EasySep Negative Selection Human B cell Enrichment Kit(Stemcell Technologies, Vancouver, BC, Canada). B cells were purifiedusing an EasySep Negative Selection Human B cell Enrichment Kit as permanufacturer's instructions (Stemcell Technologies, Vancouver, BC,Canada).

7.3 Assessment of CD40 Binding EC50 Values Using Human and Non-HumanPrimate PBMCs or B Cells

PBMCs (rhesus, cynomolgus or human) or tonsil B cells (human only) wereincubated at 4° C. for 30 min with purified labeled mAb1 or isotypecontrol antibodies in dose response (final concentration range 2.5μg/ml-0.00125 μg/ml). Cells were subsequently washed prior to beingincubated with an anti-human (non-human primate cross-reactive) and abiotinylated anti-human IgG antibody with minimal cross-reactivity toNHPs (R10; note that it was possible to distinguish membrane IgGexpressing human B cells either by including an anti-IgM stain or viadifferential intensity of FACS staining). Cells were again incubated at4° C. for 30 min prior to being washed and subject to final stainingwith streptavidin-FITC for 20 min at 4° C. Cells were subsequentlywashed and evaluation of CD40 expression on CD20+ cells was performed byflow cytometry.

7.4 Assessment of the Inhibition of CD154-Induced Proliferation of Humanand Non-Human Primate PBMCs or B Cells Seven point two-fold dilutionseries of each anti-CD40 or isotype control mAb were made in triplicatein 96 well plates in the presence of EC80 concentrations of humanrecombinant CD154 and IL-4. Starting concentrations of each anti-CD40mAb ranged from 20 μg/ml to 100 μg/ml depending on the experiment. Cellsand media controls were used as negative controls for all experiments.PBMCs (rhesus, cynomolgus or human) or tonsil B cells (human only) weresubsequently added to each well prior to incubation for 3 days at 37°C./5% CO₂. ³H-thymidine (1 μCi/50 μl/well) was added to each well forthe final 6 hours of culture prior to harvesting and determination ofthymidine incorporation using a scintillation counter.8. Efficacy of mAb1 Combined with Cyclosporine a in a KidneyAllo-Transplantation Model in Cynomolgus Monkey

8.1 Animals

All the cynomolgus monkeys (Macaca fascicularis) used were 7.5-9 yearsold males (#5529/#5533, #5523/#5524 and #5536/#5538), captive-bred,7.7±0.9 kg and originating from Philippines (Siconbrec, Makati City,Philippines). At the time of transplantation, animals presented normalhematology, serum/urine chemistry and were negative for tuberculosis,Salmonella/Shigella, for antibodies against viral agents (HerpesB,Simian T-Cell Leukemia Virus, Simian immunodeficiency virus, simian typeD retrovirus, Hepatitis B) and for relevant ecto/endoparasites. However,all animals presented antibodies against Cytomegalovirus and Hepatitis Avirus (HAV) (tested in 2010; animal #5536 was negative for HAV).Coprology from animal #5523 was tested positive for Balantidium coli inDecember 2010.

During the first week post-surgery, animals where housed in singletelemetry cages allowing visual contact with others. The rest of thetime, animals #5536/#5538 were housed together and the 4 remaininganimals were kept isolated during the whole experiment (incompatibleanimals). All animals were housed under maintained temperature (20-24°C.), at least 40% of humidity and natural light cycle. All were fed atleast twice daily with mixture of fruits and vegetables. Water and KlibaNafag 3446 pellets (Kaiseraugst, Germany) were provided ad libitum.

All experiments were performed according the Swiss Animal WelfareRegulations and under the license BS1555.

TABLE 5 Animal characteristics Species Strain Category Vendor GenderWeight Age Cynomolgus any not BioPRIM M/F 7.7 ± 0.9 7.5-9 (Macaca strainspecified fascicularis)

8.2 Experimental Conditions 8.2.1 Kidney Transplantation andPostoperative Monitoring

Donor/recipient pairs were selected according to ABO match, DRB exon2mismatch (Blancher A, Tisseyre P, Dutaur M, et al. (2006)Immunogenetics; 58(4):269-82) and responses in one-way Mixed lymphocytereaction (MLR), having MLR-stimulation indices (MLR-Sls) >7 and <47(Bigaud M, Maurer C, Vedrine, et al. (2004) J. Pharmacol. Toxicol.Methods; 50(2):153-9). The results of this selection are shown in Table6 and consisted in duo-transplant (swap transplant between 2 donors).Each recipient was implanted with a telemetric probe (Data Sciences Inc,USA) for monitoring arterial blood pressure, heart rate and motoractivity.

For surgery, general anesthesia was induced by ketamine; 10 mg/kg,intramuscularly, (i.m.) associated with atropine (0.05 mg/kg i.m.) andmaintained by ventilation with N₂O/O₂ (50:50) and propofol intravenously(i.v.) (4-10 mg/kg/h; supplemented by 5-10 mg bolus whenever required).Donor kidneys were harvested, flushed with cold (4° C.) University ofWisconsin solution (cold preservation time 4 hours) and transplantedheterotopically, using standard microvascular techniques to create anend-to-side anastomosis between the graft renal vein and the recipientvena cava and between the graft renal artery and the recipient distalaorta (anastomosis time 40 minutes). An uretero-cystoneostomy wasperformed upon appearance of urine from the graft. The native kidneyswere removed. Post-operative analgesia was provided by buprenorphine,0.01-0.02 mg/kg, i.m., three times daily); antibiotics (cefotaxime, 25mg/kg, i.m., twice daily or ceftriaxon, 50 mg/kg, i.m. in 1% lidocaine,four times daily). Analgesia and antibiotics were provided during 5days. Whenever platelet counts experienced a marked increased ordecreased, aspirin was administered at dose of 5 mg/kg i.m. daily(Aspegic, Sanofi-Aventis, Meyrin, Switzerland).

Recipients were monitored for changes in clinical and cardiovascularconditions, body weight, food and water intake (supplemented to max.100-150 ml/kg/day), urine output, hematology (using Beckmann CoulterACT5Diff), serum and urine chemistry (using a Vetscan analyzer for dailySCrea/SUrea/SAmylase determination and a Beckmann Synchron CX5 analyzerfor final confirmation of serum and urine samples). Serum creatinine(SCrea) and urea (SUrea) levels were used as markers of graft function.Transcutaneous ultrasound-guided biopsies were performed with a 16 Gneedle, as described previously (Gaschen L, Kunkler A, Menninger K, etal. (2001) Vet. Radiol. Ultrasound; 42(3):259-64), under generalanesthesia on day 30 (animals #5529 and #5533, only). In addition,special monitoring for blood coagulability and platelet aggregation wasalso performed in animals #5529/#5533 and #5523/#5524 before and aftertransplant.

Recipients were ultimately euthanized in case of (i) severe graftfailure (e.g. Screa >500 μmol/1 or Surea >20 mmol/l) associated or notwith increased ultrasound score; or (ii) general health problems and/orovert clinical signs of distress. At necropsy, the kidney allograftswere collected (including ureter and anastomosis), together with allother major organs, and processed for subsequent histological analysis.

TABLE 6 General information about the donor/recipient combinations andtreatment regimens used Body MLR-SI mAb1/CsA Recip- weight (one (mg/kg,Transplant- ient (kg) Donor ABO way) i.v./p.o.) date 5529 ♂ 7.35 5533 ♂B/B 16 30/20 21.09.10 5533 ♂ 6.1 5529 ♂ B/B 9 30/20 21.09.10 5523 ♂ 8.25524 ♂ B/B 7 30/20 18.01.11 5524 ♂ 8.3 5523 ♂ B/B 18 30/20 18.01.11 5536♂ 8 5538 ♂ B/B 47 30/20 07.06.11 5538 ♂ 8.45 5536 ♂ B/B 11 30/2007.06.118.2.2 mAb1 and Cyclosporine A (CsA) Treatments

mAb1 was provided in liquid form being freshly thawed on the day ofinfusion from −80° C. The application of 30 mg/kg/i.v. was done onceweekly, excepting for the first three doses on day −1, 0 and 1 (pre- andpost-transplant). CsA for oral administration (p.o.) was a microemulsionpreconcentrate (Sandimmun Neoral® drink solution, 100 mg/ml, NovartisPharma AG). CsA was applied at a daily dose (starting on day −1) of 20mg/kg/p.o., in combination with mAb1 (see Table 6).

8.2.3 Monitoring of mAb1 Pharmacokinetics (PK), Immunogenicity (PrimateAnti-Human Antibody) and Pharmacodynamics (PD)

Blood samples (500 μl serum) were collected (prior to i.v. dosing) forthe determination of mAb1 exposures at day −1, 3, 7 (baseline and 15min), 14, 28, 42, 56, 70, 84 and 100. For CsA determinations, bloodsamples were collected before oral dosing or CsA (C0/C24) and 2 hoursafter the application (C2). C2 corresponds to the peak levels of CsAabsorption. All the material was stored at −80° C. until furtherprocessing (CsA detection kit, Hot Star Taq Master Mix, QiagenMinnesota, US). Samples for mAb1-immunogenicity were collected (50 μlserum) on days −1, 7, 14, 28, 42, 56, 70, 84 and 100 in kept frozen −80°C. Briefly, ninety-six well microtiter plates were coated withrecombinant human CD40. These were stored at 4° C. nominal overnight.Following blocking, Calibrator standards (Cs), Quality Control (QC)samples and sample specimens containing mAb1 were added to the plate.The plate was incubated at +25° C. nominal for 120 minutes withagitation. Following washing of the plate, mouse anti human kappa lightchain antibody followed by HRP goat anti mouse (H+L) conjugate was addedto the plate to detect any mAb1 bound to the recombinant CD40. This wasvisualized by the addition of a chromogenic substrate (TMB) and theintensity of the colour produced (absorbance) was directly proportionalto the concentration of mAb1 present. The concentration of mAb1 insamples was then back-calculated from a calibration curve. PD sampleswere obtained on 2-3 days before transplant as baselines (0.5 ml inheparin). Afterwards the collection followed the same scheduled as forPK samples. CD20+ and CD3+ cells were counted with TruCount Tubes(Becton Dickinson, cat#340334) used according to the manufacturer'sinstructions with an anti-human CD40-APC mAb (Clone 5C3, BectonDickinson, cat#555591), an anti-human CD3-PerCP (Clone SP34-2, BectonDickinson, cat#552851) and of anti-human CD20-FITC mAb (Clone LT20,Immunotools, cat#21279203X2). Data was acquired on an LSRII flowcytometer (Becton Dickinson Biosciences) using DIVA (version 6.1.1)software. Lymphocytes and beads were gated in the FSC/SSC dot blotaccording to size and granularity and further analyzed for expression ofCD20 and CD3.

8.2.4 Histology

All collected tissues (graft biopsies or at necropsy) were examinedmacroscopically and fixed in 4% buffered formalin. After dehydration,they were embedded in paraffin wax. Three μm-thick sections were cutfrom paraffin blocks and stained with Hematoxylin and eosin (HE). Threeadditional stainings (Periodic acid Schiff, trichrome, and Verhoeff)were performed on kidney sections. The biopsy and necropsy samples wereexamined by an experienced pathologist and scored according to the Banff07 classification of renal allograft pathology (Solez K, Colvin R B,Racusen L C, et al (2008) Am. J. Transplant; 8(4):753-60). Peer reviewwas also performed by external experts.

In addition, immunohistochemistry for the complement protein C4d wasperformed using a polyvalent anti-C4d antibody suitable for stainingparaffin sections (Regele H, Exner M, Watschinger B, et al. 2001,Nephrol. Dial. Transplant; 16: 2058-2066). C4b is considered a stableand reliable marker of acute humoral rejection (AHR). After fixation andparaffin wax embedding, glass slides (SuperFrostPlus®, Menzel-Glaeser,Germany) with 3 μm-thick sections were prepared and dried overnight inan oven at 37° C. for optimal adhesion to the slides. Human rejectedkidney sections were added as a positive control. Before use the slideswere deparaffinized in Xylene (10 min), rehydrated through gradedethanol and placed in distilled water. Antigen retrieval was carried outby pressure-cooking for 10 min at 1 bar in citrate-buffer (pH 6.0) aspreviously described (Segerer S, Mack M, Regele H, Kerjaschki D,Schlondorff D. Kidney Int. 1999; 56: 52-64). For immunostaining, thefollowing procedure was used: (i) inactivate endogenous peroxidase with0.5% H2O2 in absolute methanol for 20 min at room temperature; (ii) washslides in 0.01M PBS, pH 7.4 (Sigma-Aldrich Chemie GmbH, Germany); (iii)incubate slides with 4% fat free powdered milk “Rapilait” in PBS,(Migros Genossenschaftsbund, Switzerland) for 60 min at roomtemperature; tap off, do not wash; (iv) incubate one slide with pAbrabbit anti-human C4d (Biomedica Medizinprodukte, Vienna, Austria) 1:40in PBS containing 1% NGtS overnight at +4° C. The second slide serves asnegative control by applying rabbit isotype control (Zymed LaboratoriesInc, USA) instead of primary antibody; (v) wash slides in 0.01M PBS, pH7.4; (vi) incubate all slides with biotinylated goat anti-rabbit IgG(Vector Laboratories, Inc. USA) 1:200 in PBS containing 1% NGtS for 30min at room temperature; (vii) wash slides in 0.01M PBS, pH 7.4; (viii)incubate with Streptavidin/HRP (Vector Laboratories, Inc. USA) in PBSfor 30 min at room temperature, (ix) development of HRP activity withAEC+(DakoCytomation Corp., Carpenteria, USA) for 8-10 min, controlstaining intensity microscopically, wash in distilled water, (x)counterstain with Dako® Automation Hematoxylin (DakoCytomation Corp.,Carpenteria, USA) for 2 min and blue in running tap water for 5 min;(xi) mount with an aqueous mounting medium (Medite Medizintechnik AG,Switzerland).

Example 1 Evaluation of the Agonistic Activity of mAb1, mAb2 and mAb3

The experimental data are based on the use of isolated, unfractionatedprimary human PBMCs. Whole PBMC preparations (instead of isolated Bcells or monocytes) more closely mimics the in vivo situation where ananti-CD40 mAb could have multiple direct and indirect effects ondifferent leukocyte cell types. Using this PBMC proliferation assay itwas determined that aglycosylated anti-CD40 mAbs mAb1 (N297A) and mAb2(D265A), which retained the amino acid sequence of the antigen bindingportion unchanged from the parental Chir12.12 antibody, retained the nonagonistic, CD40L blocking properties of the parental Chir12.12 mAb. Theantibody mAb3 (LALA mutant) was weakly agonistic in the presence ofIL-4.

In particular, the experimental results show that none of the Fc silentanti-CD40 mAbs were capable of stimulating cell division by human PBMCs(n=4 donors), a result similar to that observed with the parentalChir12.12 mAb (see FIG. 1). PBMCs proliferated in response to CD40L.Neither mAb1 or mAb2 could enhance CpG2006 or anti-IgM F(ab′)₂ inducedproliferation of PBMCs (FIG. 2). Additionally, Chir12.12 failed toenhance anti-IgM F(ab′)₂ induced proliferation of PBMCs, however unlikemAb1 and mAb2 it completely inhibited CpG induced PBMC proliferation.

In the presence of IL-4, mAb3 (LALA mutation) was observed to induce lowbut reproducible (n=4 donors) levels of thymidine incorporation abovethat induced by IL-4 alone, whereas mAb1, mAb2 and Chir12.12 did not(FIG. 3). Collectively these results indicated that with the exceptionof mAb3 (in the presence of IL-4), none of the anti-CD40 mAbs possessedagonistic activity.

The above results also clearly demonstrated that mAb1 and mAb2 did nothave agonist activity in the presence of co-stimulatory signals. This isan important finding as it is likely that the leukocytes in a patientwith chronic autoimmune disease may have an activated or partiallyactivated phenotype and thus be sensitized to signaling via CD40 orother stimuli. It was noted that Chir12.12 (but not the Fc silent mAbsor CD40L) completely inhibited CpG2006 induced PBMC proliferation.CpG2006 is a synthetic ligand for Toll-like receptor 9 (TLR9), areceptor demonstrated to bind pathogen and host derived ssDNA. In humansTLR9 is expressed by B cells and (to a lesser extent) monocytes inperipheral blood. As CpG containing ODNs have previously been shown toenhance ADCC (Moga, et al. 2008), it can be speculated that CpG2006 isable to enhance ADCC mediated by the germline IgG1 Fc portion of theparental Chir12.12 mAb.

Example 2 Evaluation of the Ability of the Fc Silent Anti-CD40 mAbs toBlock CD40L-Mediated PBMC Proliferation

Previous data indicated that Chir12.12 could block CD40L-mediatedproliferation of primary human B cells and human B cell lymphoma celllines. We measured inhibition of CD40L-mediated PBMCs proliferation byChir12.12 and the three antibodies according to the invention mAb1,mAb2, mAb3. Table 7 below presents the IC50 values for such inhibitiontabulated in mg/ml (results presented as the mean of triplicate cultureswith SEM and representative of 4 donors, independent experiments). Theresults demonstrate that Chir12.12 can also inhibit CD40L-mediated PBMCsproliferation. Additionally mAb1, mAb2 and mAb3 also completely blockedCD40L-mediated PBMC division with a potency similar to Chir12.12. Noneof the anti-CD40 mAbs blocked anti-IgM+IL-2 induced PBMC proliferation(data not shown) suggesting that the blocking activity of the anti-CD40mAbs was target dependent (and not related to Fc function).

TABLE 7 IC50 values for anti-CD40 mAb mediated inhibition ofCD40L-mediated proliferation of PBMCs (μg/ml). Antibody IC50 Chir12.12(wt Fc) 0.176 mAb1 (N297A) 0.058 mAb2 (D265A) 0.146 mAb3 (LALA) 0.118

Example 3 Binding of Anti-Human CD40 Antibody Variants to Human BJABCell Line

To exclude possible changes in specific binding to CD40, the binding ofthe three variants mAb1, mAb2 and mAb3 was tested in comparison to theparental Chir12.12 on a B cell line, BJAB, which constitutivelyexpresses CD40. Binding was tested in a dose-titration starting at 10μg/mL in a 1:2 dilution.

To compare the curves of the different experiments, the highest medianfluorescence intensity of each individual dose-titration was set to 100%binding and a non-linear curve fitting was applied. In two separateexperiments all four antibody variants Chir12.12, mAb1, mAb2 and mAb3showed equivalent binding curves on BJAB cells (FIG. 5). No changes inthe binding capabilities of the variants could be observed with thedifferent mutations of the Fc binding region of Chir12.12.

The above results thus demonstrated that the mutation of the Fc bindingsite did not impact the CD40 binding site of the variable regions of theparental Chir12.12. mAb1, mAb2 and mAb3 retained an equivalent bindingof CD40 on BJAB cells.

Example 4 Stimulation/Inhibition of TNF Alpha Release from HumanMonocytes Derived Dendritic Cells by the Anti-CD40 Fc Variants

Stimulation of TNF Alpha Release from Human MoDCs by the Anti-CD40 FcVariants

Some anti-human CD40 antibodies have been shown to have agonisticeffects on different cell population (Gruber 1989). To exclude that theFc variants mAb1, mAb2, mAb3 and Chir12.12 lead to the activation ofMoDCs, we investigated whether the antibodies on their own induce TNFαrelease when incubated for 48 h with the cells. In contrast to theantagonistic assay, seven day old human MoDCs were cultured for 48 honly in the presence of all four anti-CD40 variants in a dose-responsecurve starting at 10 μg/mL. Subsequently, supernatants were tested forthe amount of TNFα by ELISA. All anti-CD40 variants mAb1, mAb2, mAb3 andChir12.12 did not induce the release of TNFα from MoDCs as compared tothe CD40L-stimulation as a positive control (data not shown). No dosedependency could be observed by all four antibody variants. The amountsof TNFα did not rise significantly above the level of unstimulated MoDCs(Data not shown). Therefore, an agonistic activity of these fourantibodies could be excluded.

Inhibition of TNF Alpha Release from Human MoDCs by the Anti-CD40 FcVariants

The parental Chir12.12 antibody blocks the interaction of CD40-CD40L andtherefore should also block cell activation. Stimulation of CD40 onhuman monocyte-derived dendritic cells with CD40L trimers leads forexample to the release of pro-inflammatory cytokines like TNFα (Ma2009). Again, the change in the Fc region should not impact the blockingfunction of the variants in comparison to Chir12.12. All four antibodiesshould inhibit CD40L-mediated TNFα release from human MoDCs with anequivalent IC50.

Therefore, seven day old human MoDCs were stimulated for 24 h withMegaCD40L, a double trimeric, recombinant construct in the presence ofall four blocking anti-CD40 variants. Subsequently, supernatants weretested for the amount of TNFα by ELISA. All Fc-mutated variants mAb1,mAb2 and mAb3 showed the same dose-response inhibition like Chir12.12 inthree separate experiments (data not shown). Preliminary results alsoshow a similar inhibition of IL-23 release from human MoDCs (data notshown).

A non-linear curve fitting was applied to estimate an IC50 of all fourantibodies and the average IC50 from the experiments was calculated. Theaverage IC50 of the four variants for TNFα release from human MoDCsranges between 32 ng/mL and 40 ng/mL (Table 8). In summary, themutations in the Fc region did not impact the antagonistic effects ofthe antibody variants.

TABLE 8 IC50 of the different Fc variants for the inhibition of TNFalpha #1 #2 #3 Average SEM Chir12.12 n.c. 41 30 36 4 mAb1 23 44 58 40 10mAb2 41 12 42 33 10 mAb3 39 8 47 32 12 IC50 in ng/mL for the differentanti-human CD40 Fc variants from three independent experiments. Thenon-linear curve fitting in #1 for Chir12.12 did not allow a validestimate of an IC50 (n.c. = not calculated).

It was thus shown by the above results that all four variants wereinactive in inducing TNFα release from MoDCs. More importantly, allvariants mAb1, mAb2 and mAb3 inhibited CD40L-mediated cytokineproduction by human MoDCs with similar efficacy in vitro as compared toChir12.12.

Example 5 Data Summary

The following table 9 summarizes some of the important properties of theantibodies mAb1, mAb2 and mAb3 of the invention in comparison to theparental Chir12.12 antibody.

TABLE 9 Comparative data Selection Criteria Chir 12.12 mAb1 mAb2 mAb3Binding of anti- 0.55 0.69 0.49 0.33 CD40 Abs to human CD40 (Biacore, KdnM) ADCC activity 100% <1% <1% 40% (normalized specific lysis) Agonistactivity on >10000 >10000 >10000 >10000 huPBMCs (EC50, ng/ml) CD40Linhibition - 13 15 17 15 huPBMCs (IC50, ng/ml) T_(1/2) (days) - 9.3 +/−0.90 8.8 +/− 0.49 11.6 +/− 2.3 n.d Rat PK (10 mg/kg)

Example 6 Brief Description of Useful Amino Acid and NucleotideSequences for Practicing the Invention

SEQ ID NO: Description of the sequence 1 HCDR1 amino acid sequence ofCHIR-12.12, mAb1, mAb2, mAb3 2 HCDR2 amino acid sequence of CHIR-12.12,mAb1, mAb2, mAb3 3 HCDR3 amino acid sequence of CHIR-12.12, mAb1, mAb2,mAb3 4 LCDR1 amino acid sequence of CHIR-12.12, mAb1, mAb2, mAb3 5 LCDR2amino acid sequence of CHIR-12.12, mAb1, mAb2, mAb3 6 LCDR3 amino acidsequence of CHIR-12.12, mAb1, mAb2, mAb3 7 VH amino acid sequence ofCHIR-12.12, mAb1, mAb2, mAb3 8 VL amino acid sequence of CHIR-12.12,mAb1, mAb2, mAb3 9 Amino acid sequence of full length heavy chain ofCHIR-12.12 10 Amino acid sequence of full length light chain ofCHIR-12.12 11 Amino acid sequence of full length heavy chain of mAb1 12Amino acid sequence of full length light chain of mAb1 13 Amino acidsequence of full length heavy chain of mAb2 14 Amino acid sequence offull length light chain of mAb2 15 Amino acid sequence of full lengthheavy chain of mAb3 16 Amino acid sequence of full length light chain ofmAb3 17 Amino acid sequence of Fc region of mAb1 18 Amino acid sequenceof Fc region of mAb2 19 Amino acid sequence of Fc region of mAb3 20 DNAencoding Full length heavy chain of CHIR-12.12 21 DNA encoding Fulllength light chain of CHIR-12.12 22 DNA encoding Full length heavy chainof mAb1 23 DNA encoding Full length light chain of mAb1 24 DNA encodingFull length heavy chain of mAb2 25 DNA encoding Full length light chainof mAb2 26 DNA encoding Full length heavy chain of mAb3 27 DNA encodingFull length light chain of mAb3 28 Amino acid sequence of human CD40

Example 7 Useful Amino Acid and Nucleotide Sequences for Practicing theInvention

SEQ Detailed amino acid  ID NO: or nucleotide sequences  1 SYGMH  2VISYEESNRYHADSVKG  3 DGGIAAPGPDY  4 RSSQSLLYSNGYNYLD  5 LGSNRAS  6MQARQTPFT  7 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLVTVSS  8DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLISLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFGPGTKVDIR  9QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLVTVSSASTKGPSVFPLAPASKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 10DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLISLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFGPGTKVDIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 11QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYEESNRYH ADSVKGRFTI SRDNSKITLY LQMNSLRTEDTAVYYCARDGGIAAPGPDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNAKTKPREEQYASTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 12DIVMTQSPLSLTVTPGEPAS ISCRSSQSLL YSNGYNYLDWYLQKPGQSPQVLISLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGVYYCMQARQTPFTFGPGTKVD IRRTVAAPSV FIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 13QVQLVESGGGVVQPGRSLRL SCAASGFTFS SYGMHWVRQAPGKGLEWVAVISYEESNRYH ADSVKGRFTI SRDNSKITLY LQMNSLRTEDTAVYYCARDGGIAAPGPDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVAVS HEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 14DIVMTQSPLSLTVTPGEPAS ISCRSSQSLL YSNGYNYLDWYLQKPGQSPQVLISLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGVYYCMQARQTPFTFGPGTKVD IRRTVAAPSV FIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 15QVQLVESGGGVVQPGRSLRL SCAASGFTFS SYGMHWVRQAPGKGLEWVAVISYEESNRYH ADSVKGRFTI SRDNSKITLY LQMNSLRTEDTAVYYCARDGGIAAPGPDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 16DIVMTQSPLSLTVTPGEPAS ISCRSSQSLL YSNGYNYLDWYLQKPGQSPQVLISLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGVYYCMQARQTPFTFGPGTKVD IRRTVAAPSV FIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 17APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 18APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 19APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 20CAGGTGCAGTTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGAGGAAAGTAATAGATACCATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGATCACGCTGTATCTGCAAATGAACAGCCTCAGAACTGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGGGTATAGCAGCACCTGGGCCTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCAAGTACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCGCTAGCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGTCCCAGTCCAGGGCAGCAAGGCAGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAGGCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGGTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGTACCAACCTCTGTCCCTACAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 21GATATTGTGATGACTCAGTCTCCACTCTCCCTGACCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCCAGTCAGAGCCTCCTGTATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGGTCCTGATCTCTTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCGACAAACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAGACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 22CAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCCGGTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACGGCATGCACTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCGTGATCTCCTACGAGGAATCCAACAGATACCACGCTGACTCCGTGAAGGGCCGGTTCACAATCTCCCGGGACAACTCCAAGATCACCCTGTACCTGCAGATGAACTCCCTGCGGACCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGGAGGAATCGCCGCTCCTGGACCTGATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTTCCAGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACGCCTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCACAGGTGTACACACTGCCCCCCAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAAGGCTTCTACCCCTCCGATATCGCCGTGGAGTGGGAGTCCAACGGACAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCC GGCAAG 23GACATCGTGATGACCCAGTCCCCCCTGTCCCTGACCGTGACACCTGGCGAGCCTGCCTCTATCTCCTGCAGATCCTCCCAGTCCCTGCTGTACTCCAACGGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCCCCACAGGTGCTGATCTCCCTGGGCTCCAACAGAGCCTCTGGCGTGCCCGACCGGTTCTCCGGCTCTGGCTCTGGCACCGACTTCACACTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCGGCAGACCCCCTTCACCTTCGGCCCTGGCACCAAGGTGGACATCCGGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 24CAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCCGGTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACGGCATGCACTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCGTGATCTCCTACGAGGAATCCAACAGATACCACGCTGACTCCGTGAAGGGCCGGTTCACAATCTCCCGGGACAACTCCAAGATCACCCTGTACCTGCAGATGAACTCCCTGCGGACCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGGAGGAATCGCCGCTCCTGGACCTGATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTTCCAGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCACAGGTGTACACACTGCCCCCCAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAAGGCTTCTACCCCTCCGATATCGCCGTGGAGTGGGAGTCCAACGGACAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCC GGCAAG 25GACATCGTGATGACCCAGTCCCCCCTGTCCCTGACCGTGACACCTGGCGAGCCTGCCTCTATCTCCTGCAGATCCTCCCAGTCCCTGCTGTACTCCAACGGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCCCCACAGGTGCTGATCTCCCTGGGCTCCAACAGAGCCTCTGGCGTGCCCGACCGGTTCTCCGGCTCTGGCTCTGGCACCGACTTCACACTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCGGCAGACCCCCTTCACCTTCGGCCCTGGCACCAAGGTGGACATCCGGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 26CAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCCGGTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACGGCATGCACTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCGTGATCTCCTACGAGGAATCCAACAGATACCACGCTGACTCCGTGAAGGGCCGGTTCACAATCTCCCGGGACAACTCCAAGATCACCCTGTACCTGCAGATGAACTCCCTGCGGACCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGGAGGAATCGCCGCTCCTGGACCTGATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCCGGCGGCACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTCACAGTGCCTTCAAGCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCCTGCCCTGCTCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGATCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCTATCGAAAAGACAATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTGTACACCCTGCCACCCAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCC GGCAAG 27GACATCGTGATGACCCAGTCCCCCCTGTCCCTGACCGTGACACCTGGCGAGCCTGCCTCTATCTCCTGCAGATCCTCCCAGTCCCTGCTGTACTCCAACGGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCCCCACAGGTGCTGATCTCCCTGGGCTCCAACAGAGCCTCTGGCGTGCCCGACCGGTTCTCCGGCTCTGGCTCTGGCACCGACTTCACACTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCGGCAGACCCCCTTCACCTTCGGCCCTGGCACCAAGGTGGACATCCGGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 28MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ

Example 8 Toxicology Results

The primary objective of the toxicology study was to determine thetoxicity of mAb1, following once weekly intravenous administration tothe cynomolgus monkey for 5 weeks (6 test item applications). Thenon-silent (ADCC) version of this antibody (Chir12.12) was also used inorder to compare the effects of an ADCC-active antibody with theADCC-silent version (mAb1).

In addition, animals were immunized with KLH in order to evaluate theefficacy of both anti-CD40 Abs.

There were no mortalities or changes in body weights, clinical signs,and estimated food consumption attributable to treatment with mAb1 orChir12.12. Local reactions at KLH injection sites were comparable in allgroups.

Also, there were no test item-related findings in ophthalmic andcardiovascular investigations.

In hematology a slight but statistically significant decrease in thepercentage and absolute numbers of basophils was observed in mAb1- andChir12.12-treated animals: a relation to test item-treatment cannot beexcluded. Urine analysis revealed ketones in the urine of ⅕ mAb1-treatedfemales and ⅖ Chir12.12-treated females with unclear relation to dosing.In clinical chemistry a slight trend to elevated lipase concentrationswas observed for the Chir12.12-treated males as well as one femaletreated with Chir12.12, however this was considered to be of limitedtoxicological relevance.

Blood coagulation was not affected by treatment with mAb1 or Chir12.12as assessed by prothrombin time, activated partial prothrombin time, andfibrinogen. Platelet counts appeared in the normal range. P-selectin orsCD40L concentrations in plasma did not indicate platelet activation.

In Chir12.12-treated animals, blood immunophenotyping showed a prominentdecrease in CD20⁺ B-cells, which was the expected pharmacological actionof this antibody. Especially the CD20^(low)CD21⁺ B-cells (which areconsidered to be CD40^(high)) were depleted by Chir12.12. However, alsothe CD20^(high)CD21⁻ B-cells were decreased substantially especiallytowards the end of the study. Preferentially the CD20⁺CD21⁺CD27⁻ naïveB-cells were depleted by the ADCC-active antibody Chir12.12, whereas theCD20⁺CD21⁺CD27^(—) memory B-cells were hardly affected. There was alsoan approximate decrease of 50% in the absolute number of CD16⁺ NK-cellsin Chir12.12-treated animals. As expected, following treatment with mAb1(which is ADCC-silent), the prominent decrease in CD20⁺ B-cells was notobserved nor was any decrease in absolute numbers of CD16⁺ NK-cells. Theonly B-cells showing a moderate reduction during dosing were theCD20^(high) CD21⁻ B-cells. These cells are known to be macaque specificand of germinal center origin, therefore this finding is not consideredrelevant for transition to humans.

Immunophenotyping of spleen and KLH draining lymph nodes at necropsyrevealed a similar picture. There was a decrease in the relative numberof CD20⁺ B-cells in Chir12.12-treated animals, however no relevantreduction of CD20⁺ B-cells was observed for mAb1-treated animals with aslight to moderate reduction of CD21⁻ B-cells (lymph nodes both sexes,spleen males only). There were no differences in the results ofimmunophenotyping for the right and left lymph node draining the KLHinjection site.

The T-cell dependent antibody reaction (TDAR) showed that, in comparisonto the control group, no IgG and IgM response to KLH was observed in themAb1- or Chir12.12-treated animals. As blocking of B-cell activation byinhibiting CD40-CD40 ligand interaction is the intended pharmacologicalaction of mAb1, and as Chir12.12 is intended to deplete B-cells, thisfinding is not considered toxicologically relevant.

Toxicokinetic evaluation revealed that trough concentrations increasedduring the course of the study indicating accumulation of mAb1 andChir12.12. Mean trough concentrations after the 4^(th) and 5^(th) dosewere similar, indicating close-to-steady state conditions after the5^(th) dose for mAb1 and Chir12.12. Mean exposures (both genders) overthe dosing interval (AUC_(T)) after the 4^(th) and 5^(th) dose were 906and 990 h.mg/mL, respectively for mAb1, and 757 and 751 h.mg/mL,respectively for Chir12.12. The AUC_(T) values were also indicative ofclose-to-steady state conditions after the 5^(th) dose.

Macroscopic examinations did not show any evidence of target organtoxicity, and organ weights were also within the normal range for thisspecies.

Microscopically, test item-related findings of both antibodies were seenin all lymphatic organs (spleen and lymph nodes (mesenteric, mandibular,axillary, and inguinal)), where mAb1 and Chir12.12 caused completesuppression of germinal centre development in cortical B-cell areas.CD20 immunostaining of spleen and KLH draining and contralateral lymphnode tissue showed reduced size of lymphoid follicles in the spleen aswell as B-cell depletion in spleen and lymph nodes, an effect which wasmuch more pronounced in Chir12.12-treated animals compared to thetreatment with mAb1. Germinal center findings in mAb1-treated animalscorrespond to the reduction of CD21⁻ B-cells seen at immunophenotyping.CD40 immunostaining showed a significant reduction in the staining ofCD40 in lymph nodes and spleen following treatment with either mAb1 orChir12.12.

In conclusion, based on the results of this study, once weeklyintravenous administration of mAb1 or Chir12.12 for 5 weeks (6administrations) at a concentration of 100 mg/kg to male and femalecynomolgus monkeys was well tolerated. Immunophenotyping showed adepletion of B-cells in Chir12.12-treated but not mAb1-treated animalswith exception of CD21⁻ B-cells, however the TDAR showed an absence ofIgG and IgM reaction after KLH immunization in both mAb1- andChir12.12-treated animals. Histopathology revealed lack of germinalcenters in spleen and lymph nodes from mAb1- and Chir12.12-treatedanimals. The effects on B-cells are the desired pharmacologic actionsand therefore not considered to be adverse. Theno-observable-adverse-effect level (NOAEL) is considered to be at thedose of 100 mg/kg for both mAb1 and Chir12.12 under the conditions ofthis study.

Example 9 Additional In Vitro Profiling of mAb1

The binding and functional cross-reactivity of mAb1 was determinedbetween human, Rhesus and Cynomolgus leukocytes. Table 10 shows a directcomparison of the binding EC50s for mAb1 in all three species.

TABLE 10 Human and NHP cross-reactivity of mAb1. Assay Human RhesusCynomolgus CD40 binding 0.26, 0.28 0.22 +/− 0.033 0.3, 0.24 (CD20+ Bcells- (n = 6) FACS) (EC50, μg/ml) CD154 inhibition 0.058, 0.075 0.03+/− 0.017 0.015, 0.02 (hu B cells & PBMCs) (human tonsil (n = 6) (PBMCs)(IC50, μg/ml) B cells) (PBMCs)

mAb1 binds to CD20+ cells (B cells) of all three species with comparableEC50. Additionally, mAb1 could inhibit CD154+IL-4 induced proliferationof human tonsil B cells as well as PBMCs from Cynomolgus and Rhesus.Collectively these results indicated that the ability of mAb1 to bindCD40 and inhibit CD154-induced proliferation of human B cells ornon-human primate PBMCs was very similar. The availability of in vitroreceptor occupancy (RO) data and functional inhibition enabled therelationship between these two variables to be determined for eachspecies (Table 11).

TABLE 11 Relationship between mAb1 RO and functional inhibition IC₅₀ inthe functional inhibition test^(a) Corresponding in vivo RO^(b,c)(μg/mL) (%) Rhesus 0.02551 22.9 Human 0.067 43.8 ^(a)Soluble CD154 +IL4-induced proliferation in rhesus with PBMCs and in human with TonsilB cells. ^(b)assuming in vivo KD in rhesus and in human is the same asthe one computed in the PK/PD study in cyno. ^(c)assuming mAb1 is wellabove [target] the Hill-Langmuir equation is applicable.

The results indicate that approximately 2-fold less RO is required bymAb1 Rhesus PBMCs to inhibit PBMC proliferation by 50% in comparison tohuman B cells. In human, full inhibition could be obtained with ca. 75%(in vivo predicted) RO.

Example 10 Transplant Study Graft Survival

Combination treatment with mAb1 (30 mg/kg i.v.) and Cyclosporine A, 20mg/kg orally during allograft kidney transplantation resulted in asignificant prolongation of the survival of the 6 animals involved inthe study. The grafts were functional during >91*, 31, >92*, >92*, >98*and >98* days (mean: >83.6 days) in animals #5529, #5533, #5523, #5524,#5536 and #5538, respectively (* end of protocol). The survival inuntreated animals (or treated with sub-therapeutic IS-doses) ranged from7-10 days (historical data).

Animal #5533 was euthanized 31 days post-transplant, due to acute kidneyfailure and anuria. This pathology appeared after maintainedhypertensive period and anaesthesia for biopsy collection.

Monitoring Post-Transplant (a) Creatinine (SCrea) and Urea (SUrea) SerumConcentrations

SCrea was the main parameter used for the evaluation of the kidneyfunction. In all 6 animals, SCrea levels increased above the baselinelevels one day after transplant (from 81.8±14.6 to 221.5±37.1 μmol/l).Such rise in SCrea is a common feature during the first weekpost-transplant (Table 12).

TABLE 12 Changes in SCrea observed in NHP kidney allograft recipientstreated with mAb1 and CsA combination therapy at 30 and 20 mg/kg,respectively Days post sCrea level (μmol/l) transplant #5536 #5538 #5523#5524 #5529 #5533 −4 101 109 85 73 81 86 −1 106 89 80 71 95 76 0 95 10574 72 76 69 1 234 238 213 198 277 169 3 190 261 148 155 243 191 7 112189 136 141 110 110 10 111 172 193 206 102 139 14 101 166 209 165 103156 17 113 181 277 157 94 175 21 114 162 192 117 111 206 24 112 156 155124 111 191 28 95 133 119 111 100 170 31 95 139 119 106 122 629 35 103135 110 100 104 38 104 128 111 109 108 42 107 113 110 101 114 45 95 124108 115 113 49 103 118 104 106 101 52 107 129 105 111 102 56 122 103 11295 101 59 112 105 94 97 99 63 110 120 115 115 99 66 103 132 110 104 10770 101 116 91 100 105 73 103 124 105 120 109 77 103 109 98 121 105 80103 101 102 113 109 84 101 104 120 126 110 87 107 103 111 112 108 91 100112 119 112 105 94 117 133

During the first week, SUrea concentrations experienced quick risesabove the baseline measured on day 0 (4.5±1.3 mmol/l) (Table 13). Inanimals #5529/#5533 and #5536/#5538, SUrea levels were 16-20 mmol/l (day3-5), whereas in animals #5523 and #5524 were 9 or 8 mmol/l (day 7),respectively.

TABLE 13 Changes in SUrea observed in NHP kidney allograft recipientstreated with mAb1 and CsA combination therapy at 30 and 20 mg/kg,respectively Days post sUrea level (mmol/l) transplant #5536 #5538 #5523#5524 #5529 #5533 −4 7 5 4 4 8 5 −1 7 6 5 6 9 5 0 6 4 3 4 6 5 1 14 12 78 13 9 3 20 17 5 6 16 15 7 9 14 9 8 9 11 10 5 10 17 14 6 11 14 6 8 18 135 18 17 7 10 16 8 5 15 21 7 10 14 7 4 19 24 6 9 8 6 4 17 28 8 10 11 8 412 31 6 9 11 8 4 18 35 7 10 8 7 6 38 7 8 6 5 6 42 7 9 10 7 6 45 7 9 10 86 49 9 9 8 6 8 52 6 8 6 6 7 56 8 9 9 9 6 59 4 8 9 10 8 63 6 9 8 7 7 66 711 7 7 7 70 6 9 10 9 7 73 7 9 9 10 5 77 11 14 8 10 7 80 6 7 7 8 7 84 6 79 9 6 87 6 8 8 8 6 91 7 8 10 10 5 94 7 8

One week after transplant, renal function tends to normalize andSCrea/Surea become closer to baseline levels. However, animals #5533,#5523 and #5524 (but not #5529, #5536 and #5538) presented an additionalincrease in SCrea/SUrea between days 7 and 19-25 post-transplant,indicating kidney malfunction. During this period signs likepolidipsia/poliuria (#5523 and #5524), high maintained serum calcium(SCa) (#5533) or increase in graft volume (#5523 and #5524) could beobserved.

After day 20-25, animals #5529, #5523, #5524, #5536 and #5538 improvedand showed excellent kidney function until the end of the study.However, on day 31, animal #5533 displayed a pronounced increase inSCrea/SUrea levels, confirming an acute kidney failure (SCrea; 629μmol/l and SUrea; 18 mmol/l). This pathology occurred after a maintainedhypertensive period and biopsy procedure one day before euthanasia.During this period hypertensive peaks (˜170 mmgHg systolic) and ahypotensive episode (˜40 mmgHg systolic) were recorded.

(b) Serum Amylase, Lipase Concentrations, Body Weight and PlateletCounts

Serum amylase concentrations slightly increased in all animals about 1.3fold on days 1-7 after transplantation (311±53 U/L on day 0 and 418±66.8U/L on day 7 post-transplant) (Table 14). Animal #5533 presented thehighest amylase concentration observed in the study on day 1after-transplant (1050 U/L). Before transplant, there was no differencebetween amylase levels observed before the first mAb1 dose and 24 hoursafter (day −1 and day 0).

TABLE 14 Serum amylase concentrations (U/L) in kidney transplantedanimals treated with a combination of mAb1 at 30 mg/kg i.v. and CsA at20 mg/kg p.o. Time Average after-transplant amylase (U/L) STDEV Foldincrease Day 0 311 52.95 Day 7 418.2 66.8 1.34 Day 14 459.2 71.5 1.48Day 56 526.6 121.5 1.69 Day 84 460.4 66.8 1.48

Lipase serum concentrations experienced, on average, minor changes pre-and post-transplant (Table 15). Only at the end of the experimentalprotocol, a minor increase could be seen (day 84; 2.18 fold). Animal#5533 showed the highest lipase concentration measured on day 1 (284.4U/L). Lipase data from animal #5536 and #5538 was not available. Changesin amylase and lipase concentrations were similar to the levels found inother transplant-experiments using different antibodies or low molecularweight compounds (data not shown).

TABLE 15 Serum lipase concentrations (U/L) in four of the kidneytransplanted animals treated with a combination of mAb1 at 30 mg/kg i.v.and CsA at 20 mg/kg p.o. Time after-transplant Average lipase (U/L)STDEV Fold increase Day 0 12 1.3 Day 7 13.05 2.26 1.09 Day 14 12.9 1.251.08 Day 56 16.6 3.41 1.38 Day 84 26.2 21.06 2.18

A rapid and marked body weight loss could be mainly observed in animals#5529, #5533 and #5536. In all six transplanted animals, it ranged from−4 to −18% during the first 21 days following transplant. However, bodyweight loss tended to recover after that time point and towards the endof the study.

Platelet counts were normal (300-400 cells×103/μl) or increased duringthe post-transplant period 600-800 cells×103/μl. Animal #5529 receivedaspirin treatment during 3 days (day 7-9) due to quick increase inplatelet counts. Animal #5533 presented long term thrombocytosis (>1000cells×103/μl) and received aspirin treatment between days 7-26.

(c) mAb1 Blood Levels and B-Cell Counts

A reference PK/PD study and analysis was previously conducted in whichcynomolgus monkeys were given a single intravenous dose of antibody at10 mg/kg (data not shown). In this study, concentration vs. timeprofiles exhibited clear target mediated disposition (TMD), with oneanimal demonstrating a more rapid clearance as compared to anotheranimal, as a consequence of a likely higher target expression level,emphasizing the role of target expression levels in governing PK. Fromthis study, it was also established that when mAb1 serum concentrationswere above ca. 5 microg/mL, this translated into almost 100% CD40receptor occupancy.

The design of the transplant study (weekly and high mAb1 dose levels—30mg/kg, and no recovery/washout phase) did not allow for the same levelof analysis and modeling as the previously conducted PK/PD study.Nevertheless, the following observations were made (summarised in Table16); i) mAb1 was not detected in samples collected prior to first dose,ii) mAb1 was detected throughout the all dose dosing phase and, iii) inall collected samples and inter-individual trough concentrations werevariable (1200-2500 microg/mL for cynomolgus 5524, 1000-2000 microg/mLfor cynomolgus 5523 and 850-2400 microg/mL for cynomolgus 5529). Allexposure values were well above (170- to 500-fold) the concentrationneeded to obtain full receptor occupancy in cynomolgus monkey.

TABLE 16 mAb1 serum concentration for cynomolgus monkey #5533, #5529,#5523 and #5524 mAb1 (microg/mL) Time (days) Cyno_5533 Cyno_5529Cyno_5523 Cyno_5524 −1 0 0 0 0 3 591 618 1315 1384 7 682 1003 1033 11357.01 1557 1553 2452 2669 14 925 1037 1463 1744 28 654 896 1672 1965 42851 1770 1259 56 1896 1991 1283 70 2385 1641 2534 84 2191 1266 2402 912069 1066 2071

Immunogenicity testing (monkey anti-mAb1 antibodies) was also evaluatedin this study. All samples came out negative, but high mAb1 levels inthis samples, could potentially prevented their detection due to druginterferences.

A partial depletion of CD20+ cells could be observed with time in alltreated animals. Similar observations were seen in the previouslyconducted PK/PD study (data not shown).

Histology Results

Histopathological evaluation of the kidney allografts revealed no acuteand chronic rejection in grafts #5523, #5524, #5529 and #5538, andborderline changes in graft #5536 that reached end of the experiment,i.e. (results summarised in Table 17). Minimal perivascular orinterstitial infiltrates in animals #5523 #5524 and #5538, and minimalglomerular hypercellularity in animal #5529 were observed.

TABLE 17 Histology results Animal Days post- No. Sample transplantCrea/Urea Diagnosis #5529 biopsy 30 100/4 Banff: no rejection 10-0001necropsy 91 105/5 Banff: no rejection (minimal glomerularhypercellularity, minimal intimal fibrosis) Other: Lack of GC formationin secondary lymphoid organs. Cecocolitis (B. coli). No other treatmentrelated changes. #5533 biopsy 30 170/12 Banff: IA, diffuse earlyinterstitial fibrosis, tubular vacuolization 10-0002 necropsy 31 629/18Banff: other (tubular dilatation, interstitial infiltrates and fibrosis,minimal tubulitis, abscess around anastomosis, eosinophils aroundureter) Other: Lack of GC formation in secondary lymphoid organs.Cecocolitis (B. coli). No other treatment related changes. #5523 biopsyno no 11-0001 necropsy 92 119/10 Banff: no rejectio (minimalperivascular infiltrates, mucoid material in one vein)) Lack of GCformation in secondary lymphoid organs. No other treatment relatedchanges. #5524 biopsy no no 11-0002 necropsy 92 112/10 Banff: norejection (minimal multifocal interstitial infiltrates), Lack of GCformation in secondary lymphoid organs. No other treatment relatedchanges. #5536 biopsy no no 11-0002 necropsy 98 104/8 Banff: borderlinechanges (minimal multifocal interstitial infiltrates with minimal focaltubulitis), focal plasma cells Lack of GC formation in secondarylymphoid organs. No other treatment related changes. #5538 biopsy no no11-0002 necropsy 98 114/9 Banff: no rejection (minimal multifocalinterstitial infiltrates present) Lack of GC formation in secondarylymphoid organs. Unilateral focal lympho-histiocytic inflammation in thelung. No other treatment related changes.

Animal #5533 that was euthanized on day 31 post-transplant showedtubular dilatation, interstitial infiltrates and fibrosis but onlyminimal tubulitis. In addition, prominent eosinophilic infiltration invicinity of the ureter and an abscess next to anastomosis were found.All these findings indicated longstanding poor renal function butrejection could not be confirmed.

C4d immunostaining was negative in all cases.

Lack of germinal center development with or without follicular atrophywas observed in lymphoid organs in all animals. Cecocolitis caused byBalantidium coli infection was diagnosed in animals #5529 and #5533.

No other treatment related changes were encountered.

Transplant Study—Discussion

The goal of the transplant study was to assess, in a non-human primatemodel of kidney allograft rejection, the beneficial effects of mAb1 whengiven as combination therapy with sub-therapeutic dose of CyclosporineA. In addition, it was of relevance to assess the absence of sideeffects in a model where systemic inflammation is induced.

When applied as combination therapy with a subtherapeutic dose of CsA(20 mg/kg p.o.), mAb1 demonstrated efficacy in increasing the survivalof kidney allografts in NHPs. The mean graft survival was >83.6 days and5 out of 6 animals reached the end of the experimental protocol(established in 91-98 days).

Targeting CD40 using a non-agonist blocking anti-CD40 antibody (mAb1)resulted in prolongation of graft survival in kidney or islettransplanted NHPs. In addition, we could assess a better efficacy andsafety by using mAb1 (which is Fc-silent) as compared to Chir12.12previously reported (fully human monoclonal anti-CD40 antibody of theIgG1/kappa isotype with B cell depleting and co-stimulation blockingproperties).

During the whole post-transplant period, there was absence of anyrelevant clinical pathology events (e.g. minor increase inamylase/lipase levels attributed to typical impaired kidney functionafter transplant). However, animals #5333, #5523 and #5524 presentedreduced kidney function between days 7-19. This period of time istypically characterized by a recovery of SCrea/Surea levels, bloodpressure and graft volume in post-transplant animals. In these animals,signs of impaired kidney function were seen such as increasedSCrea/SUrea (all 3 animals), transitory increase in graft volume (#5523,#5524) or polydipsia/polyuria (#5523, #5524). One hypothesis could bethat those animals developed an early rejection process, which becamecontrolled after 3 weeks of mAb1 treatment. This abnormality in theearly post-transplant phase could be due to differences induced by theimmunological mode of action of a molecule (Fc-silent) targeting CD40pathway.

One animal (#5533) was euthanized on day 31 due to acute kidney failure(no rejection). This outcome was caused by an incomplete recovery of thekidney function after the transplantation procedure. The signsindicating poor kidney function were high SUrea levels (11-19 mmol/l) ormaintained high SCa concentrations (>2.8 mmol/l). The terminal graftloss was accelerated by a maintained hypertension (>140 mmHg systolic)combined with hypotension observed during anesthesia applied during thebiopsy collection procedure and high hypertensive peaks registered thenight before euthanasia (˜170 mmHg systolic) (Palmer B F (2002) N. Engl.J. Med; 347(16):1256-1261). All those events could not be attributed tomAb1 treatment and only to individual differences in the post-transplantphase.

The high efficacy of the combination therapy could be demonstrated alsohistologically. Five out of six grafts showed excellent graft quality atthe end of the experiment. Lack of germinal center development wasobserved also in a transplant experiment with Chir12.12. No othertreatment related tissue changes were observed.

One of the long-term survivor (#5529) developed cecocolitis caused byBalantidium coli. Although, the infection is common in macaques andoften asymptomatic, immunosuppression can lead to an onset of acutedisease (Schuster F L, Ramirez-Avila L, (2008) Clin. Microbiol. Rev;21(4): 626-38). In this animal no clinical signs, such as diarrhea orbody weight loss, were observed.

Regarding the B-cell counts monitoring, a partial depletion could beobserved with time in all treated animals. This partial depletion isprobably not due to active Fc-receptor mediated depletion as mAb1 is asilenced antibody, which does not bind FcR nor mediates in vitro ADCC.The partial depletion can be a mirror of the lack of germinal centers,which is observed in the histology at the end of the experiment. Thispartial depletion may be due to the lack of survival signals.

In conclusion, the results of the transplant study support the use ofmAb1 (and by extension, the other antibodies and proteins of theinvention) as valid targets for the treatment of kidney rejection in acombination therapy with an excellent safety profile. The excellentsafety profile and efficacy further support the use of the antibodies ofthe invention in the treatment of autoimmune disorders and/orinflammatory disorders, and prevention of transplant rejection mediatedby CD40L-mediated CD40 signaling on cells expressing the CD40 antigen.

SUMMARY

Anti-CD40 antibodies have not been reported to induce hemostatic eventsin patients, however elevations in pancreatic enzymes in B cell lymphomapatients receiving the anti-CD40 Ab Chir12.12 and the possible risk ofpancreatitis precludes the use of this Fc-competent anti-CD40 antibodyin chronic autoimmune disease and transplantation for safety reasons. Wetherefore generated Fc-silent IgG1 anti-CD40 antibodies (mAb1, mAb2, andmAb3) unable to mediate antibody-dependent cellular cytotoxicity (ADCC)or complement-dependent cytotoxicity (CDC) both in vitro and in vivo.mAb1 was able to prolong non-human primate renal allograft survival incombination with sub-therapeutic doses of cyclosporine. In addition,mAb1 was able to completely suppress primary and secondary antibodyresponses to immunization with a T cell-dependent antigen. Crucially,there was no evidence of hemostatic events or abnormal pancreatichistology in either the transplant or immunization study. Collectivelythese results suggest mAb1 would be a safe and efficacious therapeutic,and could be used to treat patients suffering from B lymphocyte andantigen presenting cell driven autoimmune disease or undergoingallograft transplant where CD40-CD154 interactions are involved incontributing to pathology.

1-19. (canceled)
 20. An isolated antibody or antigen-binding portionthereof directed against a target CD40 polypeptide (SEQ ID NO: 28),wherein said antibody or antigen-binding portion comprises: a. an HCDR1(SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2), HCDR3 (SEQ ID NO: 3), LCDR1 (SEQID NO: 4), LCDR2 (SEQ ID NO: 5) and LCDR3 (SEQ ID NO: 6) and b. furthercomprises an IgG1 Fc region silenced by amino acid modification ofD265A.
 21. The isolated antibody or antigen-binding portion of claim 20,wherein said antibody or antigen-binding portion exhibits <10% specificcell lysis as measured in an ADCC assay.
 22. The isolated antibody orantigen-binding portion of claim 21, wherein said antibody orantigen-binding portion exhibits <1% specific cell lysis as measured inan ADCC assay.
 23. The isolated antibody or antigen-binding portion ofclaim 20, wherein said silent IgG1 Fc region comprises an amino acidsequence of SEQ ID NO:18.
 24. The isolated antibody or antigen-bindingportion of claim 23, comprising a heavy chain variable region sequenceof SEQ ID NO: 7 and light chain variable region sequence of SEQ ID NO:8.
 25. A pharmaceutical composition, comprising the antibody orantigen-binding portion according to claim 20, in combination with atleast a pharmaceutically acceptable excipient, diluent or carrier.
 26. Aliquid pharmaceutical formulation comprising the antibody orantigen-binding portion according to claim 20 with at least a buffer.27. A method of inhibiting CD40L-mediated CD40 signaling in a subjectsaid method comprising administering to said subject the isolatedantibody or antigen-binding portion of claim 20, or a pharmaceuticalcomposition thereof, or a pharmaceutical formulation thereof.
 28. Themethod of claim 27, wherein the subject has an autoimmune orinflammatory disorder.
 29. The method of claim 28, wherein theautoimmune or inflammatory disorder is multiple sclerosis, systemiclupus erythematosus, lupus nephritis, inflammatory bowel disease,Crohn's disease, Sjogren's syndrome, myasthenia gravis, rheumatoidarthritis, atherosclerosis, transplant rejection or graft-versus hosedisease.